A Phase II study targeting amyloid-beta with 3APS in mild-to-moderate Alzheimer disease.

BACKGROUND: As a potential disease-modifying treatment for Alzheimer disease (AD), 3-amino-1-propanesulfonic acid (3APS) is a compound that binds to amyloid beta (Abeta), a toxic protein known to aggregate, leading to amyloid plaque deposition in the brain. METHODS: We assessed the safety, tolerability, and pharmacokinetic/pharmacodynamic effect of 3APS in a randomized, double-blind, placebo-controlled Phase II study in which 58 subjects with mild-to-moderate AD were randomly assigned to receive placebo or 3APS 50, 100, or 150 mg BID for 3 months. At the end of the double-blind phase, 42 of these subjects entered an open-label phase in which they received 3APS 150 mg BID for 17 months. Assessments included plasma and CSF 3APS concentrations, CSF levels of Abeta (Abeta(40) and Abeta(42)), and total tau, as well as cognitive (Alzheimer's Disease Assessment Scale-cognitive subscale, Mini-Mental State Examination) and clinical (Clinical Dementia Rating scale-Sum of Boxes) measures. RESULTS: 3APS had no significant impact on vital signs or laboratory test values. The most frequent side effects were nausea, vomiting, and diarrhea, which were intermittent and mild to moderate in severity. Seven 3APS-treated subjects discontinued because of side effects (all causalities) over the course of the study, and there were no 3APS-related serious adverse events. 3APS crossed the blood-brain barrier, and dose-dependently reduced CSF Abeta(42) levels after 3 months of treatment. There were no psychometric score differences between groups over the 3-month double-blind period. CONCLUSION: Long-term administration of 3-amino-1-propanesulfonic acid is safe, tolerated and reduces CSF Abeta(42) levels in patients with mild-to-moderate Alzheimer disease.

Functional role of local angiotensin-converting enzyme (ACE) in adrenal catecholamine secretion in vivo.

The aim of the present study was to investigate whether exogenous angiotensin I (AngI) is locally converted to angiotensin II (AngII), which in turn results in an increase in the adrenal catecholamine (CA) secretion in the adrenal gland in anesthetized dogs. Plasma CA concentrations in adrenal venous and aortic blood were determined by an HPLC-electrochemical method. Adrenal venous blood flow was measured by gravimetry. Local administration of AngI (0.0062 to 6.2 microg, 0.0096 to 9.6 microM) to the left adrenal gland resulted in significant increases in CA output in a dose-dependent manner. Following administration of 0.62 microg (0.96 microM) of AngI, adrenal epinephrine and norepinephrine outputs increased from 20.8+/-13.6 to 250.9+/-96.4 ng x min(-1) x g(-1) (p<0.05, n = 5) and from 2.8+/-1.7 to 29.6+/-11.1 ng x min(-1) x g(-1) (p<0.05, n = 5), respectively. From the same left adrenal gland, the output of AngII increased from -0.02+/-0.04 to 26.39+/-11.38 ng x min(-1) x g(-1) (p<0.05, n = 5), while plasma concentrations of AngII in aortic blood remained unchanged. In dogs receiving captopril (12.5 microg, 0.5 mM) 10 min prior to AngI, the net amounts of CA and AngII secreted during the first 3 min after AngI were diminished by about 80% (p<0.05, n = 5) compared with those obtained from the control group. There was a close correlation (r2 = 0.91, n = 6) between the net increases in AngII and CA outputs induced by AngI. The results indicate that the local angiotensin converting enzyme is functionally involved in regional AngII formation in the canine adrenal gland in vivo. The study suggests that AngII thus generated may play a role in the local regulation of adrenal CA secretion.

Functional involvement of angiotensin AT2 receptor in adrenal catecholamine secretion in vivo.

The aim of the present study was to analyse modulations of adrenal catecholamine secretion from the adrenal gland of anesthetized dogs in response to locally administered angiotensin II (AngII) in the presence of either PD 123319 or CGP 42112, both of which are highly specific and selective ligands to angiotensin AT2 receptor. Plasma concentrations of epinephrine and norepinephrine in adrenal venous and aortic blood were quantified by a high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) method. Adrenal venous blood flow was measured by gravimetry. Local administration of AngII (0.05 microg, 0.1 microM) to the left adrenal gland increased adrenal gland catecholamine output more than 30 times that found in nonstimulated states. Administration of either PD 123319 (0.085 microg (0.23 microM) to 8.5 microg (23 microM)) or CGP 42112 (0.005 microg (0.01 microM) to 5 microg (10 microM)) did not affect the basal catecholamine output significantly. The increase in adrenal catecholamine output in response to AngII was inhibited by approximately 80% following the largest dose of PD 123319. CGP 42112 significantly attenuated the catecholamine response to AngII by approximately 70%. PD 123319 and CGP 42112 were devoid of any agonist actions with respect to catecholamine output by the adrenal gland in vivo. Furthermore, both PD 123319 and CGP 42112 inhibited the increase in adrenal catecholamine secretion induced by local administration of AngII. The present study suggests that AT2 receptors play a role in mediating catecholamine secretion by the adrenal medulla in response to AngII receptor agonist administration in vivo.

Functional evidence for L-type Ca2+ channels controlling ANG II-induced adrenal catecholamine release in vivo.

The aim of the present study was to investigate the functional involvement of L- and/or N-type Ca2+ channels in adrenal catecholamine secretion in response to exogenous angiotensin II (ANG II) in anesthetized dogs. Plasma catecholamine concentrations in adrenal venous and aortic blood were determined by a high-performance liquid chromatography-electrochemical method. In the first series of experiments, repeated infusions of BAY K 8644 locally into the left adrenal gland at 15-min intervals resulted in significant and reproducible increases in adrenal catecholamine secretion. Nifedipine, similarly administered 5 min before BAY K 8644, diminished BAY K 8644-induced catecholamine secretion in a dose-dependent manner and completely blocked the catecholamine response at the highest dose tested. In the second series of experiments, local infusion of ANG II resulted in a significant increase in adrenal catecholamine secretion. The maximum catecholamine response to ANG II was attenuated by approximately 65% in the presence of nifedipine at the dose that abolished the BAY K 8644-induced catecholamine release. This inhibition by nifedipine remained unchanged in the presence of omega-conotoxin. The present study shows that dihydropyridine-sensitive L-type Ca2+ channels are operative in the adrenal medulla of the dog in vivo. The results indicate that the L-type Ca2+ channels are only partially implicated in the local regulation of ANG II-induced adrenal catecholamine secretion, suggesting the existence of another mechanism. However, omega-conotoxin-sensitive N-type Ca2+ channels are unlikely to be functionally involved in postsynaptic mechanisms mediating adrenal catecholamine secretion in response to exogenous ANG II under in vivo conditions.

Inhibition by BMS 186295, a selective nonpeptide AT1 antagonist, of adrenal catecholamine release induced by angiotensin II in the dog in vivo.

The aim of the present study was to investigate whether a novel nonpeptide AT1 selective antagonist, BMS 186295 (BMS), can antagonize adrenal catecholamine release induced by local administration of angiotensin II (AII) in anesthetized dogs. Plasma catecholamine concentrations in adrenal venous and aortic blood were determined by an HPLC-electrochemical method. AII was locally administered to the left adrenal gland in the absence and presence of BMS. In the first group (n = 7), local infusion (0.5 mL/min, 1 min) of AII (0.001-1.0 micrograms/mL) resulted in a significant dose-dependent increase in the basal secretion of adrenal catecholamines. Aortic catecholamine levels and mean aortic pressure remained unchanged at all doses tested. In the second group (control, n = 10), four repeated infusions (at intervals of 15 min) of AII at 0.1 micrograms/mL resulted in significant increases of adrenal catecholamine secretion compared with the baseline. In the third group receiving BMS given locally to the gland (n = 8), the basal adrenal catecholamine secretion was not significantly altered by BMS itself at any dose tested. However, the net catecholamine response to AII (0.1 micrograms/mL) was significantly and dose dependently attenuated by approximately 40, 60, and 80% in the presence of BMS at doses of 0.1, 1.0, and 10 micrograms/mL, respectively, compared with the control group. The study indicates that BMS dose dependently blocks AII-induced catecholamine secretion in the dog adrenal gland in vivo.

Simultaneous evaluation of medullary secretory functions of normal and acutely denervated adrenals.

Adrenal medullary secretory function of the right innervated gland was simultaneously compared with that of contralateral acutely denervated gland in anesthetized dogs. During bilateral carotid artery occlusion (BCO), output (in ng/min) from right innervated gland of epinephrine and norepinephrine increased from 86.6 +/- 33.0 and 34.4 +/- 15.1 to 280.8 +/- 86.7 (P less than 0.01, n = 7) and 104.4 +/- 40.6 (P less than 0.01, n = 7), respectively. By contrast, epinephrine output from left denervated gland increased only slightly (P less than 0.05), and norepinephrine did not increase significantly. Net catecholamine output from left denervated gland was markedly attenuated by approximately 90% (P less than 0.01, n = 7) compared with that from right innervated gland. During BCO in the second group of dogs, catecholamine output from sham-denervated left gland increased significantly (P less than 0.01, n = 7) to an extent slightly lower than that observed in right innervated gland. In the third group, intravenous injections of dimethylphenylpiperazinium (5 and 15 micrograms/kg) resulted in a dose-dependent increase (P less than 0.05, n = 7) in catecholamine output from both right innervated and left denervated gland. The results indicate that the present procedure of acute surgical adrenal denervation can eliminate the centrally mediated adrenal response, whereas the medullary secretory response to blood-borne substances remains intact. This model may be a useful tool for studying neuronal and humoral medullary secretory functions in vivo under various experimentally induced stress conditions.

Effect of functional adrenalectomy on glucagon secretion and circulating catecholamines during insulin hypoglycemia in the dog.

The present study was carried out to determine whether an increase in the pancreatic immunoreactive glucagon (IRG) secretion during the acute phase of insulin-induced hypoglycemia depends on circulating catecholamines of adrenal origin. Hypoglycemia was induced by a bolus insulin injection (0.15 IU/kg, i.v.) in dogs anesthetized with sodium pentobarbital (35 mg/kg, i.v.). Plasma aortic epinephrine (E) and norepinephrine (NE) concentrations increased significantly 30 min after the injection of insulin. At this time point, a functional adrenalectomy (diversion of bilateral adrenal venous blood from the systemic circulation) was performed for 5 min. The increased aortic E and NE concentrations significantly decreased reaching, within 5 min, a level below the corresponding preinjection control value. The basal output of pancreatic IRG (6.58 +/- 1.12 ng/min, n = 6) significantly increased (24.93 +/- 2.77 ng/min, p less than 0.05, n = 6) 30 min after insulin injection. During the functional adrenalectomy, the increased pancreatic IRG output diminished rapidly, within 5 min, to approximately 50% (11.73 +/- 3.19 ng/min, p less than 0.05, n = 6) of the value observed 30 min after insulin administration. In the other group of dogs receiving sham adrenalectomy, the increased aortic E and NE concentrations and pancreatic IRG output following insulin injection remained elevated above the levels observed immediately before the sham adrenalectomy. The net decrease in IRG output during the adrenalectomy was significant (p less than 0.05) compared with the corresponding net IRG output observed in the sham group.(ABSTRACT TRUNCATED AT 250 WORDS)

Pancreatic glucagon secretion during a short period of hemorrhage in anesthetized dogs.

Glucagon has been implicated in the hormonal metabolic response to hemorrhage. However, evidence for this has been obtained largely from observations of circulating plasma glucagon concentration. A clear increase in the pancreatic glucagon secretion remains to be demonstrated. Plasma concentrations of pancreatic immunoreactive glucagon (IRG) and insulin (IRI) were determined in portal venous and aortic blood, and plasma glucose in aortic blood. Dogs were bled (approximately 15 mL/kg) until aortic systolic blood pressure dropped to approximately 50% (70.5 +/- 8.1 mmHg, n = 7) (1 mmHg = 133.32 Pa) of its control value (135 +/- 7.1 mmHg, n = 7), and the hemorrhagic hypotension was maintained for 10 min. The net portal venous IRG delivery rate rose significantly and continued to increase during the hemorrhagic hypotension despite a significant fall in the portal venous blood flow. Aortic IRG increased significantly along with the increase in portal venous IRG delivery rate (r = 0.838, n = 42, p less than 0.01). The portal venous delivery rate of IRI decreased significantly in response to hemorrhage. The aortic IRG/IRI concentration ratio increased significantly during the hemorrhage-induced hypotension. Aortic glucose concentration increased significantly 5 min after hemorrhage and continued to rise until the end of the hemorrhagic hypotension. The present study demonstrates that the secretion of pancreatic glucagon actually increases during the early phase of hemorrhage. The results also indicate that the increase in aortic IRG during the hemorrhagic hypotension is due to the increased pancreatic glucagon secretion. It is suggested that the pancreatic glucagon may be involved in the early hyperglycemic response to hemorrhage.

Alpha 2-adrenoceptor modulation of catecholamine and neuropeptide Y responses during haemorrhagic hypotension in anaesthetized dogs.

The acute effects of oxymetazoline, an alpha 2-adrenoceptor agonist, and idazoxan, an alpha 2-adrenoceptor antagonist, on the release of neuropeptide Y were evaluated during haemorrhage in pentobarbital-anaesthetized dogs. Plasma concentrations of neuropeptide Y and catecholamines (adrenaline, noradrenaline, and dopamine) were determined in samples simultaneously collected from aorta, portal vein, and adrenal veins. In control dogs, adrenal catecholamine output, aortic concentrations neuropeptide Y and catecholamines markedly increased during the hypotension period. However, adrenal neuropeptide Y output decreased significantly during this period. Portal venous noradrenaline and neuropeptide Y concentrations increased significantly. In dogs treated with idazoxan, catecholamine output from the adrenals increased to an extent similar to that observed in control dogs. However, the increase in noradrenaline and neuropeptide Y in aortic or portal venous blood during haemorrhage was significantly potentiated in the presence of idazoxan. Administration of oxymetazoline abolished this increase, but did not alter adrenal catecholamine or neuropeptide Y output. The present study demonstrates that neuropeptide Y is co-released with noradrenaline from sympathetic nerve fibers during haemorrhage. Since the release of neuropeptide Y appeared to follow a similar time course to that of noradrenaline release, the present observations suggest that haemorrhagic hypotension enhances both neuropeptide Y and noradrenaline release presumably through a common releasing mechanism. These results also indicate that, in peripheral sympathetic nerves but not in the adrenal gland, neuropeptide Y release is also modulated presynaptically by the inhibitory alpha 2-adrenoceptors in conjunction with the noradrenaline release.

Corelease of neuropeptide Y like immunoreactivity with catecholamines from the adrenal gland during splanchnic nerve stimulation in anesthetized dogs.

The release of neuropeptide Y like immunoreactivity (NPY-li) from the adrenal gland was studied in relation to the secretion of catecholamines (CA: NE, norepinephrine; E, epinephrine) during the left splanchnic nerve stimulation in thiopental-chloralose anesthetized dogs (n = 16). Plasma concentrations of NE, E, and NPY-li were determined in the left adrenal venous and aortic blood. Adrenal outputs of NPY-li, NE, and E were 2.4 +/- 0.4, 1.4 +/- 0.2, and 7.3 +/- 1.7 ng/min, under basal conditions, respectively. These values increased significantly (p less than 0.05; n = 8) in response to a continuous stepwise stimulation at frequencies of 1, 3, and 10 Hz given at 3-min intervals during 9 min, reaching a maximum output of 4.6 +/- 0.9 (NPY-li), 240.2 +/- 50.2 (NE), and 1412.5 +/- 309.7 ng/min (E) at a frequency of 10 Hz. Burst electrical stimulation at 40 Hz for 1 s at 10-s intervals for a period of 10 min produced similar increases (p less than 0.05) in the release of NPY-li (4.8 +/- 1.0 ng/min, n = 8), NE (283.5 +/- 144.3 ng/min, n = 8), and E (1133.5 +/- 430.6 ng/min, n = 8). Adrenal NPY-li output was significantly correlated with adrenal NE output (r = 0.606; n = 24; p less than 0.05) and adrenal E output (r = 0.640; n = 24; p less than 0.05) in dogs receiving the burst stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of glucagon in hyperglycemic response during a short period of hemorrhage in anesthetized dogs.

A role of pancreatic glucagon in hemorrhage induced hyperglycemia was studied in anesthetized dogs with or without functional adrenalectomy (ADRX), surgical hepatic denervation (HNX), and surgical pancreatectomy (PCX). Plasma epinephrine, norepinephrine, and glucose concentrations were determined in both hepatic venous and aortic blood. Plasma glucagon (IRG) and insulin (IRI) levels were determined in aortic blood. All dogs were bled until aortic systolic pressure dropped to approximately 50% (64.8 +/- 1.6 mmHg, n = 25) of its control value (136.7 +/- 4.4 mmHg, n = 25), and the hypotension was maintained for 5 min. In control dogs (n = 10), hemorrhage markedly increased aortic epinephrine and hepatic venous norepinephrine. Similarly, aortic IRG, hepatic venous glucose and aortic glucose rose significantly during hemorrhage. In dogs with HNX combined with ADRX (n = 10), aortic epinephrine and hepatic venous norepinephrine remained unchanged during hemorrhage. Aortic IRG concentration, however, increased to a level similar to that observed in the control group. Aortic glucose increased significantly along with significant increases in hepatic venous glucose. In dogs with PCX combined with HNX and ADRX (n = 5), the increases in aortic IRG, hepatic venous glucose and aortic glucose observed in the first two groups in response to hemorrhage were virtually abolished. The results indicate that the increase in aortic IRG during hemorrhage is of pancreatic origin. We conclude that the pancreatic glucagon may be involved in the hyperglycemic response to hemorrhage, most likely through glucose mobilization by the liver during the early phase of hemorrhagic hypotension.

Plasma catecholamine and glucose concentrations during hemorrhagic hypotension in anesthetized dogs.

Hemorrhage-induced hepatic glycogenolytic responses were compared in pentobarbital-anesthetized dogs with a temporary functional adrenalectomy (ADRX) and in dogs with an acute hepatic denervation (HNX). Plasma concentrations of catecholamines [CAs; epinephrine (E), norepinephrine (NE), and dopamine (DA)] were determined in aortic (AO), hepatic venous (HV), portal venous (PV), and adrenal venous (ADV) blood collected simultaneously before, during, and after hemorrhage (H). Plasma glucose (GL) concentrations were measured in AO and HV blood. AO blood was bled (5.3 +/- 0.5 ml.kg-1.min-1, n = 42) until AO systolic pressure dropped to half (72.9 +/- 4.7 mmHg) of its control value (148.0 +/- 4.4 mmHg), and the hypotension was maintained for 5 min. In control dogs (CTL; n = 12), H markedly increased ADV CAs with a predominant increase in E over NE and DA. AO CAs increased similarly. By contrast, however, changes in HV CAs were characterized by a significant increase in NE that was more pronounced than the increases in E and DA. The increases in NE were associated with significant increases in both HV GL and AO GL. In dogs with ADRX (n = 10), AO CAs remained unchanged during H, but both HV NE and HV GL rose concomitantly to an extent similar to that observed in CTL dogs. In dogs with HNX (n = 10), HV NE remained unchanged during H, but HV GL increased to an extent similar to that observed in dogs with ADRX, along with significant increases in AO CAs. In dogs with HNX combined with ADRX (n = 10), HV NE and AO CAs did not change at all during H, resulting in a marked attenuation (50%, P less than 0.05) of the increasing response of HV GL.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct evidence that an increase in aortic norepinephrine level in response to insulin-induced hypoglycemia is due to increased adrenal norepinephrine output.

This study reports on the major source of circulating norepinephrine that is known to increase, progressively, during sustained hypoglycemia induced by intravenous insulin administration. Plasma concentrations of epinephrine, norepinephrine, and dopamine were simultaneously determined for adrenal venous and aortic blood in dogs anesthetized with sodium pentobarbital. The model used allowed us to perform a functional adrenalectomy (ADRX), while continuously monitoring the adrenal medullary secretory function. Under basal conditions, the net output (micrograms/min) of adrenal epinephrine, norepinephrine, and dopamine were 0.169 +/- 0.074, 0.067 +/- 0.023, and 0.011 +/- 0.003, respectively. Plasma concentrations (ng/mL) of aortic epinephrine, norepinephrine, and dopamine were 0.132 +/- 0.047, 0.268 +/- 0.034, and 0.034 +/- 0.009. Following insulin injection (0.15 IU/kg, i.v.), the net output (micrograms/min) of adrenal epinephrine, norepinephrine, and dopamine increased gradually (p less than 0.05), reaching the values of 0.918 +/- 0.200, 0.365 +/- 0.058, and 0.034 +/- 0.007 30 min after insulin administration. Similarly, aortic epinephrine, norepinephrine, and dopamine concentrations (ng/mL) increased significantly (p less than 0.05) to 0.702 +/- 0.144, 0.526 +/- 0.093, and 0.066 +/- 0.024. The aortic glucose concentration (mg/dL) was diminished from 81.8 +/- 4.1 to 36.9 +/- 3.4 (p less than 0.01). After taking the blood sample at 30 min following insulin administration, ADRX was immediately performed. Five minutes after the onset of ADRX, the net output (micrograms/min) of adrenal epinephrine, norepinephrine, and dopamine increased further to 1.707 +/- 0.374 (p less than 0.05), 0.668 +/- 0.139 (p less than 0.05), and 0.052 +/- 0.017.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of adrenal norepinephrine output to increase aortic norepinephrine during carotid sinus reflex activation in anesthetized dogs.

Changes in circulating plasma catecholamine (CA: E, epinephrine; NE, norepinephrine; and DA, dopamine) concentrations in aortic (AO) blood were investigated in relation to variable rates of CA secretion from both adrenal (ADR) glands in response to bilateral carotid artery occlusion (BLCO) in vagotomized dogs anesthetized with sodium pentobarbital. During BLCO (3 min), AO systolic pressure (AP) increased along with significant increases in ADR-CA output, renal venous (RV) CA output, as well as in AO-E and NE concentrations. A ratio of NE:E in ADR venous and AO blood did not exceed 0.42 +/- 0.09 and 1.09 +/- 0.24 upon BLCO, respectively. In contrast, the NE:E ratio in RV blood increased significantly from 5.39 +/- 0.91 to 9.78 +/- 1.31. Following adrenalectomy (ADRX), the increase in AO-NE in response to BLCO was significantly attenuated by approximately 56%, but the increase in RV-NE output was not affected by ADRX. The results show that in vagotomized dogs, NE is co-released with E from the adrenal glands upon BLCO. The data also indicate that the increase in AO-NE concentration was dependent to a similar extent on the simultaneous increases in ADR-NE output and neuronal NE release. We conclude that under conditions where the sympathoadrenal system is activated, circulating plasma NE concentration may be significantly affected by an increase in ADR-NE output. Sympathetic neuronal contributions would, thereby, be overestimated in assessing overall sympathetic nerve activity by measuring circulating NE. NE concentrations in local venous effluent from individual organs may be more reliable estimates of the sympathetic nerve activity.

High-performance liquid chromatographic determination of the lecithin/sphingomyelin ratio in amniotic fluid.

A high-performance liquid chromatographic (HPLC) procedure has been developed for the separation of phospholipids commonly found in amniotic fluid. The chromatographic separation was achieved on a 25-cm column packed with LiChrosorb DIOL (10 mum). A 3-cm column packed with silica was fitted between the injector and the DIOL column to provide complete separation of lecithin (L) and sphingomyelin (S) from the remaining amniotic fluid phospholipids. The eluted phospholipids were quantitated employing an ultraviolet absorption detector set at 203 nm. The new HPLC separation described herein has improved the resolution and peak sharpness of L and S. Furthermore, phosphatidyl glycerol and phosphatidyl inositol were completely separated and quantitated. Amniotic fluid L/S ratios determined by this technique have been compared to those of an established thin-layer chromatographic procedure.

Miniature two-dimensional thin-layer chromatographic separation of lecithin and sphingomyelin.

A miniature two-dimensional thin-layer chromatographic procedure employing silica gel impregnated glass-microfiber chromatography sheets (commercial product, ITLC-type SG sheets) has been developed for the separation of lecithin (L) and sphingomyelin (S) from a standard lipid mixture containing L, S, lysolecithin, phosphatidyl inositol (PI), phosphatidyl serine (PS), phosphatidyl ethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol. The newly developed procedure eliminates possible interference from PI and PS. Complete separation of L and S was easily achieved with chromatographic solvent migration times of approximately 3 and 2 min for the first and second dimensions, respectively. The lipids were visualized by charring and fluorescent staining techniques. The procedure has been adapted for the separation of L and S from amniotic fluid samples.