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Choline acetyltransferase- and substance P-like immunoreactive elements in fetal striatal grafts in the rat: a correlated light and electron microscopic study.

Fetal striatal neurons were transplanted into the ibotenic acid-lesioned rat striatum. Three months after transplantation, the graft tissue was processed for choline acetyltransferase- and substance P-like immunoreactivity and was subsequently examined at the light and electron microscopic levels. The study demonstrated that choline acetyltransferase- and substance P-like-immunoreactive neurons were homogenously present throughout fetal striatal grafts, although in decreased numbers compared with those in the normal rat striatum. The majority of the choline acetyltransferase-immunoreactive neurons had fusiform, oval, or polygonal somata with somatic diameters greater than 20 microns and contained deeply invaginated nuclei surrounded by copious cytoplasm. In addition, choline acetyltransferase-immunoreactive neurons with somatic diameters between 10 and 20 microns were also demonstrated. The grafts' substance P-like-immunoreactive neurons, which had somatic diameters between 10 and 25 microns and had oval or polygonal perikarya, could be classified into two types based on their ultrastructural characteristics. Type I neurons contained an unindented nucleus which was surrounded by a thin rim or moderate amount of cytoplasm, whereas Type II immunoreactive neurons contained an indented nucleus which was surrounded by copious cytoplasm. Choline acetyltransferase- and substance P-like-immunoreactive dendrites in the grafts' neuropil were contacted by multiple unlabeled axon terminals. In addition, choline acetyltransferase- and substance P-like-immunoreactive axon terminals forming symmetric contacts with unlabeled dendrites were present within the graft. The study demonstrated that many of the neuroanatomical features of choline acetyltransferase- and substance P-like-immunoreactive elements found in the normal rat striatum are present in mature fetal striatal grafts.

Animals↗

Choline blockage of currents through Ca(2+)-activated K+ channels in bovine chromaffin cells.

The action of choline on "maxi" Ca(2+)-activated K+ channels was studied in excised patches of bovine chromaffin cell membranes. Choline (20-70 mM) applied to the internal surface of the membrane reduced the single channel current amplitudes, which can be explained by a fast channel block. The block is concentration- and voltage-dependent and is rapidly and completely reversed upon washout. The block becomes progressively greater with depolarization. The estimates of blocking parameters vary from channel to channel but appear to fall in two groups. A larger group (two-thirds of cases) with moderate affinity [KD(0) = 88.5 mM] and low voltage dependence (delta = 0.26) and a smaller group (one-third of cases) with very low affinity (KD = 306 mM) and moderate voltage dependence (delta = 0.59). The open state probability appears not to be affected at any choline concentration (up to 70 mM) or membrane potential (from -20 to +60 mV) studied, suggesting that choline does not affect the channel gating kinetics. Since the affinity of the choline block is low to moderate, the intracellular choline is not expected to alter the current flow through "maxi" Ca(2+)-activated K+ channels unless the choline concentration close to the protoplasmic membrane is much higher than the mean cellular concentration.

Adrenal Medulla↗

Choline acetyltransferase and NADPH diaphorase are co-expressed in rat spinal cord neurons.

Phenotypic diversity underlies the complex functioning of the nervous system. One characteristic in which neurons differ from one another is the kind of molecules that they use for intercellular signalling. The classical neurotransmitter acetylcholine, synthesized by the enzyme choline acetyltransferase, is used by five groups of neurons in the rat spinal cord. Another messenger is nitric oxide, which is synthesized by nitric oxide synthase. Neurons that express nitric oxide synthase can be stained specifically by NADPH diaphorase histochemistry. In the spinal cord, approximately five groups of neurons are labeled by the diaphorase reaction, and some of these populations overlap with cholinergic groups. To determine the proportions of neurons that co-express choline acetyltransferase and nitric oxide synthase, we performed choline acetyltransferase immunocytochemistry and diaphorase histochemistry on single sections of rat spinal cord. Some cell types were single-labeled: somatic motor neurons were choline acetyltransferase-immunoreactive only, and neurons in lamina II were diaphorase-positive only. Four cell groups included double-labeled cells. Autonomic motor neurons were either double-labeled (62%) or choline acetyltransferase-only (37%), partition cells in lamina VII were double-labeled (54%) or choline acetyltransferase-only (45%), neurons in laminae III-V of the dorsal horn were double-labeled (70%) or diaphorase-only (27%), and neurons surrounding the central canal were double-labeled (56%), choline acetyltransferase-only (23%) or diaphorase-only (21%). These data indicate that certain spinal cord populations may be heterogeneous with regard to the intercellular messenger phenotypes involving acetylcholine and nitric oxide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

The characterization of a sodium-dependent high affinity choline uptake system unassociated with acetylcholine biosynthesis.

The cardiac ganglion of the horseshoe crab, Limulus polyphemus, was incubated in Chao's solution containing 0.01 microM [3H]choline at room temperature (25 +/- 2 degrees C) and the ganglion readily accumulated the radiolabel. The ganglion uptake of [3H]choline was linear over 60 min. Kinetic analysis revealed dual choline uptake systems within the cardiac ganglion, a high affinity uptake system (Km = 2.2 microM, Vmax = 0.16 pmoles/mg/min) and a low affinity system (Km = 92.3 microM, Vmax = 3.08 pmoles/mg/min). The high affinity uptake system was sodium-dependent and inhibited by micromolar concentrations of hemicholinium-3. A 15 min pre-exposure of the ganglion to Chao's solution containing 90 mM potassium stimulated a significant increase in choline uptake. There was no detectable synthesis of [3H]acetylcholine from the [3H]choline taken up by the cardiac ganglion. The major portion of the extractable label appeared in a fraction which co-electrophoresed with phosphorylcholine. These results suggest that the sodium-dependent high affinity [3H]choline uptake system of the cardiac ganglion subserves a specific requirement for choline which is unrelated to a cholinergic function.

Acetylcholine↗

Hemicholinium-3 derivatives A-4 and A-5 affect choline and acetylcholine metabolism.

The neuroblastoma-glioma hybrid cell (NG108-15) has a sodium-dependent, high-affinity choline transport system with a Km of 16.0 +/- 3.4 microM and a Vmax of 214.5 +/- 27.7 pmol/min/mg protein. A-4, A-5 and HC-3 produce dose-dependent inhibition of high-affinity choline transport in NG108-15 cells. Following 24 h exposure to approximately the EC50 of each inhibitor, no significant decrease was found in total choline accumulation or in choline incorporation into phosphatidylcholine. However, when additional inhibitor was added during the 24 h incubation, significant decreases in choline accumulation were produced by A-4 and A-5. Following 24 h exposure to each compound, only A-4 was able to significantly affect free choline content. In contrast, each inhibitor was able to significantly decrease acetylcholine content following 24 h exposure. Possible reasons for consistent decreases in acetylcholine versus minimal changes in choline metabolism will be discussed.

Acetylcholine↗

Insights into molecular plasticity of choline binding proteins (pneumococcal surface proteins) by SAXS.

Phosphocholine moieties decorating the pneumococcal surface are used as a docking station for a family of modular proteins, the so-called choline binding proteins or CBPs. Choline recognition is essential for CBPs function and may also be a determinant for their quaternary structure. There is little knowledge about modular arrangement or oligomeric structures in this family. Therefore, we have used the small angle X-ray scattering (SAXS) technique combined with analytical ultracentrifugation in order to model the three-dimensional envelope of two highly different CBPs: the phage encoded Cpl-1 lysozyme and the pneumococcal phosphorylcholine esterase Pce. Both enzymes have an N-terminal catalytic module and a C-terminal choline-binding module (CBM) that attaches them to the bacterial surface and comprises six and ten sequence repeats in Cpl-1 and Pce, respectively. SAXS experiments have shown an inherent conformational plasticity in Cpl-1 that accounts for the different relative position of these regions in the solution and crystal structures. Dimerization of Cpl-1 upon choline binding has been also visualised for the first time, and monomer-monomer interactions take place through the first CBR where a non-canonical choline binding site has now been identified. This mode of association seems to be independent of the absence or presence of the Cpl-1 catalytic module and reveals that the arrangement of the monomers differs from that previously found in the isolated CBM dimer of pneumococcal LytA amidase. In contrast, Pce displays the same modular disposition in the solution and crystal structures, and remains almost invariant upon choline binding. The present results suggest that protein dimerization and duplication of CBRs may be alternative but not equivalent ways of improving cell wall recognition by CBPs, since they provide different interaction geometries for choline residues present in (lipo)teichoic acids.

Binding Sites↗

The fetal origins of memory: the role of dietary choline in optimal brain development.

Fetal nutrition sets the stage for organ function in later life. In this review we discuss the fetal and neonatal origins of brain function. Numerous research observations point to the importance of choline for the developing fetus and neonate. This essential nutrient is involved in 1-carbon metabolism and is the precursor for many important compounds, including phospholipids, acetylcholine, and the methyl donor betaine. Dietary intake of choline by the pregnant mother and later by the infant directly affects brain development and results in permanent changes in brain function. In rodents, perinatal supplementation of choline enhances memory and learning functions, changes that endure across the lifespan. Conversely, choline deficiency during these sensitive periods results in memory and cognitive deficits that also persist. Furthermore, recent studies suggest that perinatal choline supplementation can reduce the behavioral effects of prenatal stress and the cognitive effects of prenatal alcohol exposure in offspring. The likely mechanism for these effects of choline involves DNA methylation, altered gene expression, and associated changes in stem cell proliferation and differentiation. The currently available animal data on choline and hippocampal development are compelling, but studies are needed to determine whether the same is true in humans.

Brain↗

Contribution of 11C-choline positron emission tomography/computerized tomography to preoperative staging of advanced transitional cell carcinoma.

PURPOSE: Current imaging modalities for preoperative staging of advanced transitional cell carcinoma of the bladder or upper urinary tract are not sensitive for detection of metastases. This study examines the contribution of 11C-choline positron emission tomography/computerized tomography to preoperative staging of transitional cell carcinoma. MATERIALS AND METHODS: We prospectively evaluated 18 patients with 19 advanced transitional cell carcinomas (17 bladder tumors and 2 upper tract transitional cell carcinomas). All patients had computerized tomography of the chest, abdomen and pelvis negative for metastases. 11C-choline positron emission tomography/computerized tomography was performed on a Discovery ST(R) positron emission tomography/computerized tomography system. Finally 16 patients underwent radical surgery and positron emission tomography/computerized tomography images were compared to histopathological findings. Two patients were not operated on due to the findings on 11C-choline positron emission tomography/computerized tomography. RESULTS: 11C-choline uptake was found in all primary transitional cell carcinomas, with a maximum standardized uptake value of 7.3 +/- 3.2 (mean +/- SD). The series included 3 patients with refractory bladder carcinoma in situ, which was visualized in all 3, with a standardized uptake value of 6.9 +/- 5.6. In 6 patients uptake of 11C-choline in lymph nodes as small as 5 mm was visualized (standardized uptake value 3.8 +/- 1.4). Of these patients 4 underwent surgery and histopathology confirmed malignancy in 3 of 4. No additional patients with positive lymph nodes were found on histopathology. Metastases were visualized in bones with normal architecture on computerized tomography in 4 patients (standardized uptake value 5.2 +/- 1.1) and were confirmed by followup computerized tomography. CONCLUSIONS: In this small series 11C-choline positron emission tomography/computerized tomography was highly sensitive for primary and metastatic transitional cell carcinoma. Carcinoma in situ, lymph node metastases and early bony metastases were visualized. 11C-choline positron emission tomography/computerized tomography is a promising tool for preoperative staging of advanced transitional cell carcinoma.

Aged↗

Perinatal choline supplementation does not mitigate motor coordination deficits associated with neonatal alcohol exposure in rats.

Prenatal alcohol exposure can disrupt brain development, leading to a variety of behavioral alterations including learning deficits, hyperactivity, and motor dysfunction. We have been investigating the possibility that perinatal choline supplementation may effectively reduce the severity of alcohol's adverse effects on behavioral development. We previously reported that perinatal choline supplementation can ameliorate alcohol-induced learning deficits and hyperactivity in rats exposed to alcohol during development. The present study examined whether perinatal choline supplementation could also reduce the severity of motor deficits induced by alcohol exposure during the third trimester equivalent brain growth spurt. Male neonatal rats were assigned to one of three treatment groups. One group was exposed to alcohol (6.6 g/kg/day) from postnatal days (PD) 4 to 9 via an artificial rearing procedure. Artificially and normally reared control groups were included. One half of subjects from each treatment received daily subcutaneous injections of a choline chloride solution from PD 4 to 30, whereas the other half received saline vehicle injections. On PD 35-37, subjects were tested on a parallel bar motor task, which requires both balance and fine motor coordination. Ethanol-exposed subjects exhibited significant motor impairments compared to both control groups whose performance did not differ significantly from one another. Perinatal choline treatment did not affect motor performance in either ethanol or control subjects. These data indicate that the beneficial effects of perinatal choline supplementation in ethanol-treated subjects are task specific and suggest that choline is more effective in mitigating cognitive deficits compared to motor deficits associated with developmental alcohol exposure.

Analysis of Variance↗

Effect of hypoxia on the uptake of [methyl-3H]choline, [1-14C] acetate and [18F]FDG in cultured prostate cancer cells.

INTRODUCTION: Choline, acetate and glucose ([2-(18)F]fluoro-2-deoxyglucose, [(18)F]FDG) analogs are under investigation as positron emission tomography (PET) tracers for the imaging of prostate cancer; however, their response to tumor hypoxia has not been clarified. METHODS: The uptake of [methyl-(3)H]choline, [1-(14)C]acetate and [(18)F]FDG was monitored in androgen-independent PC-3 cells and androgen-sensitive LNCaP cells under aerobic or anoxic conditions. The effect of androgen depletion was also examined. RESULTS: PC-3 cells exhibited aerobic choline and acetate uptake five to six times higher than FDG uptake, whereas LNCaP cells showed choline uptake 2.2-fold higher than acetate uptake and 10-fold higher than FDG uptake. After 4 h of anoxia, PC-3 cells showed an 85% increase in FDG uptake, a 15% decrease in choline uptake and a 36% increase in acetate uptake, whereas LNCaP cells showed a 212% increase in FDG uptake, a 28% decrease in choline uptake and no change in acetate uptake. Androgen depletion resulted in a marked decrease in the uptake of all tracers in LNCaP cells but no changes in PC-3 cells. CONCLUSION: In aerobic conditions, both PC-3 and LNCaP cells exhibited an order of uptake preference as follows: choline>acetate>FDG. In hypoxic cells, the order is reversed, reflecting diverse biochemical responses to hypoxia. These findings may help to explain PET imaging findings of the diverse responses of these tracers in different stages and locations of prostate cancer. Androgen depletion markedly suppressed the uptake of all three tracers in LNCaP cells, which suggests the potential for underestimation of the disease state when PET imaging is performed subsequent to antiandrogen therapy.

Acetates↗

Gut bacteria associated with an atherogenic TMAO-dietary pattern and choline-rich foods among aging women.

BACKGROUND AND AIMS: Choline can be metabolized by gut bacteria with a choline utilization gene, CutC, as identified through genome sequencing studies. This metabolism produces trimethylamine, the precursor to the atherosclerotic metabolite trimethylamine N-oxide (TMAO). Bacterial species involved in trimethylamine production in free-living humans have been under-investigated. We previously developed the TMAO dietary pattern (TMAO-DP), which is predictive of plasma TMAO and choline. We evaluated associations between the TMAO-DP, dietary choline, and choline-rich foods (fish, red meat, eggs) with the abundance of species with CutC. We also explored associations between the TMAO-DP and microbiome diversity. METHODS AND RESULTS: This cross-sectional analysis included 287 women (mean age = 79.6 years) from the Women's Health Initiative. Diet was assessed using a food frequency questionnaire. Stool samples were collected and the V3-V4 regions of the 16S ribosomal RNA were sequenced. Adjusted linear regression models evaluated associations between the TMAO-DP with the CLR-transformed abundance of species with CutC and with alpha-diversity indices. For beta-diversity, PERMANOVA examined measures of Aitchison distance within and between quartiles of the TMAO-DP. Associations between dietary choline and choline-rich foods with the abundance of species were evaluated using linear regression. The TMAO-DP was associated with Acidaminococcus intestini [Beta (SE): 0.23 (0.09), p-value = 0.035] and Desulfovibrio desulfuricans [Beta (SE): 0.16 (0.6), p = 0.035]. The TMAO-DP was not associated with alpha- or beta-diversity. CONCLUSION: This study provides evidence that Desulfovibrio desulfuricans and Acidaminococcus intestini, two species identified as having CutC by gene sequencing, may produce trimethylamine from diet in free-living women.

Humans↗

Prognostic implications of elevated whole blood choline levels in acute coronary syndromes.

Troponins I and T represent the current biomarker standard for diagnosis of myocardial infarction. Even small increases of cardiac troponins have prognostic implications, but not all patients at risk are correctly classified, particularly at admission. We identified elevated whole-blood choline as a promising marker and performed a prospective study of 327 patients with a suspected acute coronary syndrome that focused on the analysis of troponin-negative patients. Diagnostic classification of patients and the definition of troponin cutoffs were performed according to the new European Society of Cardiology/American College of Cardiology criteria. Blood was sampled serially and choline was measured using high-performance liquid chromatography mass spectrometry in whole blood. Patients were followed for 30 days. In patients with negative troponin I test results at admission (n = 250), choline was a predictor of cardiac death and nonfatal cardiac arrest (hazard ratio 6.0, p = 0.003), life-threatening arrhythmias (hazard ratio 3.75, p = 0.004), heart failure (hazard ratio 2.87, p = 0.002), and coronary angioplasty (hazard ratio 2.57, p = 0.001). In multivariate analysis of troponin-negative patients, choline was the strongest predictor of cardiac death or arrest (odds ratio 6.05, p = 0.01). Choline was not a marker for myocardial necrosis but indicated high-risk unstable angina in patients without acute myocardial infarction (sensitivity 86.4%, specificity 86.2%). Thus, an increased concentration of choline at hospital admission is a predictor of adverse cardiac events in patients with suspected acute coronary syndromes. Whole blood choline may be useful for early risk stratification of these patients, particularly if troponin results are negative on admission.

Angina, Unstable↗

Choline kinase from yeast.

Choline kinase, the initial enzyme of the CDP-choline pathway, mediates the conversion of choline to phosphorylcholine and is localized in the supernatant fraction of cells. The enzyme also catalyzes the phosphorylation of ethanolamine, functioning as the initial enzyme of the CDP-ethanolamine pathway as well. Yeast choline kinase is encoded by a single structural gene, CKI, which was cloned by the genetic complementation of the choline kinase mutation cki. The deduced sequence comprises 582 amino acid residues with a molecular mass of 66316 Da and bears local sequence similarity to various protein kinases and bacterial antibiotic phosphotransferases. The expression of yeast choline kinase is transcriptionally repressed by myo-inositol and choline in a coordinate manner with other phospholipid-synthesizing enzymes in yeast.

Amino Acid Sequence↗

Choline rise in the rat hippocampus induced by electroconvulsive shock treatment.

BACKGROUND: Human hippocampal choline decreases in major depression episodes. This decrease was recently measured by 1H magnetic resonance spectroscopy (MRS), and it has been found that its level normalizes during antidepressive electroconvulsive therapy. We hypothesized a hippocampal choline increase in the rat brain under electroconvulsive shock (ECS) treatment. METHODS: Rat hippocampi (n = 28) were investigated via magnetic resonance spectroscopy and signal intensities of choline (Cho), total creatine (tCr), and N-acetyl aspartate (NAA) were measured and expressed as ratios before and after six ECS treatments. RESULTS: After ECS treatment, hippocampal choline increases significantly: Cho/tCr ratio: +13% and Cho/NAA ratio: +19% increase. CONCLUSIONS: We found a rise of relative choline concentration induced by ECS treatment in rat hippocampus measured in vivo with magnetic resonance spectroscopy. This increase corresponds to the increase of choline in human hippocampus after electroconvulsive shock treatment. Because choline measured via 1H-spectroscopy is believed to represent primarily phosphocholine and glycerophosphocholine, and therefore phospholipase A2 activity and membrane turnover, our results are in good agreement with reported ECS-induced hippocampal mossy fiber sprouting, increased synaptic plasticity, and neurogenesis.

Animals↗

Transport of choline in rat brain slices.

Enzyme-modified amperometric microsensors have been utilized in the investigation of acetylcholine and choline diffusion in solution and choline uptake and diffusion in rat brains. A small amount of the substance of interest was introduced by pressure injection and transport to the sensor was monitored. The apparent diffusion coefficients for acetylcholine and choline in agarose gel perfused with physiological solutions were determined to be 5.2 +/- 0.7 x 10(-6) cm2/s and 6.1 +/- 0.8 x 10(-6) cm2/s, respectively. Choline transport was monitored in two brain regions: the caudate and anterior hypothalamus. The transport time of choline in the caudate was concentration dependent, but was unaffected by the presence of a competitive, high-affinity uptake inhibitor, hemicholinium-3. The apparent diffusion coefficient (D) and uptake rate (k) for choline in the caudate and anterior hypothalamus were calculated using a model for point source diffusion coupled with first-order uptake kinetics. The effect of the sensors' response time on the measurements was removed by deconvolution. The D and k were 1.8 +/- 0.1 x 10(-6) cm2/s and 2.0 +/- 0.1 x 10(-2) s-1 in the caudate and 1.9 +/- 0.1 x 10(-6) cm2/s and 3.2 +/- 0.6 x 10(-2) s-1 in the anterior hypothalamus. The reduced diffusion coefficient determined in brain tissue compared to agar gel is consistent with the increased tortuosity of the brain microenvironment. A substance in brain tissue, presumably acetylcholinesterase, prevents the use of differential measurements of acetylcholine because choline sensors became sensitive to acetylcholine.

Acetylcholine↗

Potentiation and inhibition of neuronal alpha4beta4 nicotinic acetylcholine receptors by choline.

The effects of choline on alpha4beta4 nicotinic acetylcholine receptors, expressed in Xenopus oocytes, were investigated using in the two-microelectrode voltage clamp technique. Particular attention was paid to the interaction between the effects of acetylcholine and choline. Choline was a low-affinity agonist of alpha4beta4 receptors with an efficacy of 10% as compared to acetylcholine. Responses evoked by 1 microM acetylcholine were potentiated by low concentrations of choline and inhibited by > 10mM choline, resulting in a bell-shaped concentration-effect relationship. Conversely, the effects of choline on responses evoked by 300 microM acetylcholine resulted in a monophasic inhibition curve with an IC(50) of 0.87 mM. The data were fitted by a two-site receptor occupation model, which accounts for similar effects of various cholinergic ligands on heteromeric nicotinic receptors. The results indicate that the potentiation was a competitive effect, whereas the inhibition was due to a mixture of competitive and non-competitive effects. It is concluded that choline acts as a potent, endogenous co-agonist at heteromeric alpha4beta4 nicotinic receptors.

Acetylcholine↗

The effects of choline on body temperature in conscious rats.

Choline (75-300 microg) produced dose-dependent hypothermia when injected intracerebroventricularly (i.c.v.). Pre-treatment with the muscarinic receptor antagonist, atropine (10 microg, i.c.v.), blocked the hypothermic effect of choline (150 microg), but the response was only partially attenuated by pre-treatment with the nicotinic receptor antagonist, mecamylamine (20 microg, i.c.v.). Pirenzepine (25 microg), a muscarinic M1 receptor antagonist, or hexahydro-siladifenidol (HHSD) (100 microg), a muscarinic M3 receptor antagonist, also blocked choline-induced hypothermia when injected centrally. Unlike the other muscarinic receptor antagonists, M2-selective 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyri do[2,3-b][1,4]benzodiazepin-6-one (AF-DX116) (10 microg), did not affect choline-induced hypothermia. We also found that choline-induced hypothermia was very sensitive to the ambient temperature. Similar to its effect at room temperature, choline produced dose-dependent hypothermia at 4 degrees C, but this effect was abolished at 32 degrees C. These data suggest that choline produces hypothermia and this effect is mediated by muscarinic receptors.

Acetylcholine↗

Histochemical technique to detect choline-containing lipids.

An almost specific and highly sensitive technique to detect histochemically choline-containing lipids is proposed. This technique is based on the effect of Hg++ and phosphomolybdic ion on the choline, that yields insoluble complexes able to react with diphenylcarbazide producing a deep blue or violet stain. The specificity and the sensitivity of this technique was investigated on filter paper strips loaded with choline-containing lipids, choline-free lipids, steroids, vitamins, proteins or choline hydrochloride. The results show that the choline-containing substances react positively. The choline-free substances react negatively except calciferol and vitamin A that show weakly positive results. This technique used on tissue sections appears very suitable to histochemical purposes.

Adrenal Glands↗