ABSTRACT
Niemann-Pick disease type C (NPC) is characterized by an accumulation of unesterified cholesterol in the endosomal/lysosomal (E/L) system, resulting in progressive neurodegeneration and death during early childhood. To investigate the cellular pathomechanism of nervous system involvement in NPC, continuous neural cell lines are desirable. In this study, we obtained neuronal and Schwann cell cultures and established spontaneously immortalized Schwann cell lines from dorsal root ganglia and peripheral nerves of NPC model mouse (spm/spm). One of the cell lines, designated SPMS9, had distinct Schwann cell phenotypes and was maintained over 10 months without phenotypic alterations. The level of Npc1 mRNA was markedly decreased, and NPC1 protein was not detectable in SPMS9 cells. These cells contained intracytoplasmic granules positive for filipin cholesterol staining and immunoreactive for GM2 ganglioside. Electron-microscopically, intracytoplasmic polymorphous membranous inclusions and vacuoles were demonstrated in SPMS9 cells. The treatment with an inhibitor of ceramide-specific glucosyltransferase, N-butyldeoxynojirimysin (NB-DNJ) markedly reduced the intracytoplasmic granular immunofluorescence for GM2 ganglioside in SPMS9 cells, whereas the amount of filipin-positive granules remained unchanged. The SPMS9 cells retained vesicular fluorescence of cationic dye acriflavine 16-24 hours after loading, indicating the defect of transmembrane efflux pump activity of NPC1 in the E/L compartment in these cells. These immortalized Schwann cells can be useful in studies on the nervous system lesions in NPC.
Subject(s)
Niemann-Pick Diseases/pathology , Schwann Cells/pathology , Animals , Blotting, Western , Cells, Cultured , Cytological Techniques , Fluorescent Antibody Technique, Indirect , Ganglia, Spinal/pathology , Mice , Niemann-Pick Diseases/metabolism , Peripheral Nerves/pathology , Phenotype , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Schwann Cells/metabolismABSTRACT
It remains controversial whether deficiency of the Niemann-Pick C1 (npc1) protein results in altered cholesterol signaling at the endoplasmic reticulum (ER). In this report, we have measured the processed, nuclear form of sterol regulatory element binding protein (SREBP)-1 in livers of npc1 wild-type, heterozygous, and homozygous deficient mice, alone, and in combination with deficiencies of the low density lipoprotein receptor (LDLR) or the multiple drug resistant (mdr)1a, P-glycoprotein. Cleavage of SREBPs to activated forms normally occurs when the ER is deficient in cholesterol. A large decrease in processed SREBP-1 was evident in fasted npc1(-/-) mice and npc1(-/-), mdr1a(-/-) mice, with no decrease evident in npc1(-/-), LDLR(-/-) mice. These results suggest that the increase in cellular cholesterol which occurs in npc1(-/-) and in npc1(-/-), mdr1a(-/-) mice includes the sites responsible for cholesterol signaling, while the similar increase in cholesterol found in npc1(-/-), LDLR(-/-) mice does not.
Subject(s)
CCAAT-Enhancer-Binding Proteins/metabolism , Cholesterol/metabolism , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum/metabolism , Proteins/metabolism , Receptors, LDL/deficiency , Transcription Factors , ATP Binding Cassette Transporter, Subfamily B/deficiency , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP-Binding Cassette Transporters/genetics , Animals , Female , Heterozygote , Homozygote , Intracellular Signaling Peptides and Proteins , Liver/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Mutant Strains , Niemann-Pick C1 Protein , Proteins/genetics , Receptors, LDL/genetics , Sex Factors , Signal Transduction/physiology , Sterol Regulatory Element Binding Protein 1ABSTRACT
Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol in most tissues and progressive neurodegeneration with the formation of neurofibrillary tangles. Neurofibrillary tangles are composed of paired helical filaments (PHF), a major component of which is the hyperphosphorylated tau. In this study we used NPC heterozygous and NPC homozygous mouse brains to investigate the molecular mechanism responsible for tauopathy in NPC. Immunoblot analysis using anti-tau antibodies (Tau-1, PHF-1, AT-180, and AT-100) revealed site-specific phosphorylation of tau at Ser-396 and Ser-404 in the brains of NPC homozygous mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases such as glycogen synthase kinase-3beta and cyclin-dependent kinase 5 were inactive. Morphological examination demonstrated that a number of neurons, the perikarya of which strongly immunostained with PHF-1, exhibited polymorphorous cytoplasmic inclusion bodies and multi-concentric lamellar-like bodies. Importantly, the accumulation of intracellular cholesterol in NPC mouse brains was determined to be a function of age. From these results we conclude that abnormal cholesterol metabolism due to the genetic mutation in NPC1 may be responsible for activation of the mitogen-activated protein kinase-signaling pathway and site-specific phosphorylation of tau in vivo, leading to tauopathy in NPC.
Subject(s)
MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , tau Proteins/metabolism , Age Factors , Alkaline Phosphatase/metabolism , Animals , Brain/enzymology , Brain/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Nucleus/metabolism , Cerebellum/enzymology , Cholesterol/metabolism , Cyclin-Dependent Kinase 5 , Cyclin-Dependent Kinases/metabolism , Cytoplasm/metabolism , Disease Models, Animal , Enzyme Activation , Glycogen Synthase Kinase 3 , Glycogen Synthase Kinases , Heterozygote , Homozygote , Hot Temperature , Immunoblotting , Lipid Metabolism , Liver/enzymology , Mice , Mice, Inbred BALB C , Microscopy, Electron , Mutation , Niemann-Pick Diseases/genetics , Phosphorylation , Purkinje Cells/metabolism , Serine/chemistry , Signal Transduction , Telencephalon/enzymology , Time FactorsABSTRACT
Niemann-Pick type C (NPC) is a neurodegenerative disorder characterized by greatly altered somatic cholesterol metabolism. The NPC1 gene has recently been cloned and shown to have sequence homology to other sterol-sensing proteins. We have used a mouse model with a disrupted npc1 gene to study the effects of the cholesterol-mobilizing compound, 2-hydroxypropyl-beta-cyclodextrins (HPBCD), on the clinical course of this disorder. Treatment with two HPBCDs, with varying levels of 2-hydroxypropyl substitution, had effects in delaying neurological symptoms and in decreasing liver cholesterol storage while a third HPBCD was without effect. The ameliorating effect was not improved by longer exposure times (commencement of exposure in utero), however, it is not known if there is transplacental transfer of HPBCDs. The combination of HPBCD with probucol or nifedipine (which have previously been shown to lower liver cholesterol in this animal model) markedly decreased liver storage of unesterified cholesterol without altering the depressed levels of esterified cholesterol. The slight effects of the HPBCDs on neurological symptoms may be partially due to their apparent non-permeation of the blood-brain barrier (BBB). This non-permeation was assayed with radioactive tracers and was also present in the mdr1a knockout mice which have greatly increased BBB permeability for many drugs. Intrathecal delivery of HPBCD by an Alzet osmotic minipump did not improve its efficacy in ameliorating neurological symptoms.
Subject(s)
Anticholesteremic Agents/therapeutic use , Cyclodextrins/therapeutic use , Niemann-Pick Diseases/drug therapy , beta-Cyclodextrins , 2-Hydroxypropyl-beta-cyclodextrin , Animals , Anticholesteremic Agents/administration & dosage , Anticholesteremic Agents/pharmacokinetics , Ataxia/drug therapy , Ataxia/physiopathology , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/physiology , Brain/drug effects , Brain/metabolism , Cholesterol/analysis , Cyclodextrins/administration & dosage , Cyclodextrins/pharmacokinetics , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Therapy, Combination , Female , Injections, Intraperitoneal , Injections, Spinal , Intracellular Signaling Peptides and Proteins , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Knockout , Niemann-Pick C1 Protein , Niemann-Pick Diseases/genetics , Niemann-Pick Diseases/physiopathology , Nifedipine/therapeutic use , Probucol/therapeutic use , Proteins/genetics , Tremor/drug therapy , Tremor/physiopathologyABSTRACT
Mouse Niemann-Pick disease type C1 (npc1), formerly designated spm (sphingomyelinosis), is an autosomal recessive lipid storage disorder. We generated a high-resolution linkage map in the 2.24-cM npc1 critical region by typing eight polymorphic markers in 2322 meioses (948 of these were previously reported). A minimal set of overlapping yeast artificial chromosomes (YACs) had previously been assembled (Hsu and Erickson 2000). The YAC 313-B-8, which covered this whole region, has been used to construct cosmid libraries. Three cosmid contigs were built, and one of them contained the npc1 locus. Two (CA)(n) microsatellites were identified, and the one new one was characterized, from the YAC-derived cosmids. The most proximal cosmid contig overlaps with markers near twirler (Tw). Both the physical map and genetic linkage map have been integrated to study the recombination frequencies in this particular region of the mouse genome, and recombination suppression due to the heterozygous insertion of DNA was suggested.
Subject(s)
Chromosome Mapping , Proteins/genetics , Recombination, Genetic/genetics , Retroelements , Animals , Chromosomes, Artificial, Yeast , Contig Mapping , Cosmids , Dinucleotide Repeats , Female , Intracellular Signaling Peptides and Proteins , Male , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Microsatellite Repeats , Mutagenesis, Insertional , Mutation , Niemann-Pick C1 Protein , Physical Chromosome Mapping , Polymorphism, Genetic , Sequence Tagged SitesABSTRACT
Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol and other lipids in the lysosomal compartment. In this report, we use subcellular fractionation and microscopy to determine the localization of the murine Niemann-Pick C1 (NPC1) protein. Fractionation of mouse liver homogenates indicates that some NPC1 cosediments with lysosome-associated membrane protein 1 (LAMP1)-containing membranes. However, a significant amount of NPC1 is also found in membranes that do not contain LAMP1. Moreover, fractionation of liver membranes and fibroblasts in the presence of a nonionic detergent showed that a fraction of NPC1 cosediments with caveolin-1 in rafts. Immunofluorescence microscopy of cultured mouse fibroblasts showed that NPC1 is found in two morphologically distinct structures. The first population is characterized by large punctate structures that do not colocalize with major organelle protein markers, but do colocalize with filipin and a small fraction of caveolin-1. Examination of these large NPC1-containing compartments using electron microscopy shows that these structures contain extensive internal membranes. The second population is represented by smaller, more diffuse structures, a fraction of which colocalize with LAMP1-positive compartments. Incubation of fibroblasts with low density lipoprotein (LDL) increases colocalization of NPC1 with LAMP1-containing compartments. This colocalization can be further enhanced by treating fibroblasts with progesterone or chloroquine. The results indicate that NPC1 is associated with an unique vesicular compartment enriched with cholesterol and containing caveolin-1, and that NPC1 cycles to LAMP1-positive compartments, presumably to facilitate the processing of LDL-derived cholesterol.
Subject(s)
Niemann-Pick Diseases/metabolism , Proteins/metabolism , Amino Acid Sequence , Animals , Antibodies , Cell Compartmentation , Cell Fractionation , Centrifugation, Density Gradient , Cholesterol/metabolism , Female , Fibroblasts/metabolism , Intracellular Signaling Peptides and Proteins , Liver/metabolism , Liver/ultrastructure , Male , Mice , Mice, Inbred BALB C , Mice, Mutant Strains , Microscopy, Fluorescence , Microscopy, Immunoelectron , Molecular Sequence Data , Niemann-Pick C1 Protein , Niemann-Pick Diseases/genetics , Proteins/genetics , Proteins/immunology , RabbitsABSTRACT
Niemann-Pick type C (NPC) is a neurodegenerative disorder with somatically altered cholesterol metabolism. The NPC1 gene has recently been cloned and shown to have sequences shared with known sterol-sensing proteins. We have used a mouse model with a disrupted Npc1 gene to study two cholesterol-lowering drugs (nifedipine and probucol) and the effects of introducing a null mutation in the low-density lipoprotein receptor (LDLR). Although these treatments significantly ameliorated liver cholesterol storage, little effect on the onset of neurological symptoms was found.
Subject(s)
Cholesterol/metabolism , Niemann-Pick Diseases/drug therapy , Nifedipine/therapeutic use , Probucol/therapeutic use , Receptors, LDL/physiology , Animals , Blood-Brain Barrier , Mice , Mice, Inbred BALB C , Mice, Knockout , Niemann-Pick Diseases/geneticsABSTRACT
This study assesses the developmental expression of the Niemann-Pick type C mRNA in vivo and in vitro in rat cerebellum. NPC is an autosomal recessive neurovisceral lipid storage disease associated with an alteration in cholesterol trafficking. In the mouse model of NPC and in the early onset form of human NPC, Purkinje neurons are among the first neurological targets, suffering stunted growth during postnatal development and dying, leading to ataxia. Recently, the genes responsible for human (NPC1) and mouse (Npc1) NPC disease have been cloned. Based on a highly homologous domain, we designed primers to look for levels of Npc1 mRNA with a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) approach using cyclophilin as an internal standard. Total RNA was isolated from various postnatal developmental stages of the rat cerebellum as template for the analyses. Npc1 transcripts were observed at postnatal day 0 and at later stages of development, both in vivo and in vitro from primary cerebellar cultures. To identify the location of Npc1 inside the cerebellum, we performed immunostaining with an anti-Npc1 antibody in primary rat cerebellar cultures identifying reactive Purkinje neurons by double-labeling with the Purkinje specific marker calbindin and sub-populations of glial cells. In summary, Npc1 is expressed in rat cerebellum in vivo and in vitro and is expressed during early postnatal development as well as in the adult cerebellum. Since Npc1 is expressed at similar levels throughout development, the vulnerability of Purkinje neurons to this disease is likely to involve disruption of an interaction with other developmentally-regulated proteins.
Subject(s)
Cerebellum/metabolism , Gene Expression Regulation/physiology , Niemann-Pick Diseases/genetics , Amino Acid Sequence , Animals , Base Sequence , Calbindins , Cells, Cultured , Humans , Immunohistochemistry , Membrane Glycoproteins/genetics , Mice , Mice, Neurologic Mutants , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Niemann-Pick C1 Protein , Niemann-Pick Diseases/pathology , Peptidylprolyl Isomerase/genetics , Purkinje Cells/metabolism , Purkinje Cells/pathology , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , S100 Calcium Binding Protein G/genetics , Sequence Homology, Amino AcidABSTRACT
We have previously demonstrated (1) an increased expression of caveolin-1 in murine heterozygous and homozygous Niemann-Pick type C (NPC) livers, and (2) an increased concentration of unesterified cholesterol in a detergent insoluble caveolae-enriched fraction from homozygous livers. To define further the relationship between caveolin-1 function and the cholesterol trafficking defect in NPC, we examined the expression and distribution of additional caveolar and signal transduction proteins. The expression of annexin II was significantly increased in homozygous liver homogenates and the Triton X-100 insoluble floating fraction (TIFF). Phosphoamino acid analysis of caveolin-1 and annexin II from the homozygous TIFF demonstrated an increase in serine and tyrosine phosphorylation, respectively. To determine the basis for increased phosphorylation of these proteins, the expression and distribution of several protein kinases was examined. The expression of PKCalpha, PKCzeta and pp60-src (protein kinases) were significantly increased in both heterozygous and homozygous liver homogenates, while PKCdelta was increased only in homozygous livers. Of the protein kinases analyzed, only CK IIalpha was significantly enriched in the heterozygous TIFF. Finally, the concentration of diacylglycerol in the homozygous TIFF was significantly increased and this elevation may modulate PKC distribution and function. These results provide additional evidence for involvement of a caveolin-1 containing cellular fraction in the pathophysiology of NPC and also suggest that the Npc1 gene product may directly or indirectly, regulate the expression and distribution of signaling molecules.
Subject(s)
Annexin A2/metabolism , Caveolins , Liver/metabolism , Membrane Proteins/metabolism , Protein Kinases/metabolism , Proteins/genetics , Animals , Annexin A2/biosynthesis , Caveolin 1 , Cholesterol/analysis , Diglycerides/analysis , Disease Models, Animal , Female , In Vitro Techniques , Intracellular Signaling Peptides and Proteins , Male , Membrane Proteins/biosynthesis , Mice , Mice, Inbred BALB C , Niemann-Pick C1 Protein , Niemann-Pick Diseases/genetics , Octoxynol , Phospholipids/analysis , Phosphorylation , Protein Kinases/biosynthesis , Signal Transduction/genetics , SolubilityABSTRACT
Niemann-Pick type C (NPC) is an autosomal recessive disease characterized by impaired cholesterol homeostasis due to a defect in the intracellular transport of unesterified cholesterol. While accumulation of lysosomal cholesterol is the most apparent microscopic finding, cholesterol has also been shown to accumulate in the trans-cisternae of the Golgi apparatus. Caveolin-1, a cholesterol-binding protein that cycles between the Golgi apparatus and the plasma membrane, has been hypothesized to participate in cholesterol transport. Using the BALB/c murine model for NPC disease, we found that the expression of caveolin-1 in total liver homogenates from heterozygous and homozygous affected animals was altered. Immunoblot analysis of liver homogenates from heterozygous mice revealed that caveolin-1 is significantly (p < 0.005) elevated, 4.9 fold, compared to normal mice. Total liver homogenates from homozygous affected mice also had a significant (p < 0.05) increase in caveolin-1 expression, 1.6 fold, compared to normal mice. Immunohistochemical staining of liver cross-sections revealed that the increased caveolin-1 was localized to sinusoidal endothelial cells in heterozygous mice. The Triton insoluble floating fraction (TIFF) was isolated using liver from each genotype and analyzed for caveolin-1 expression. Caveolin-1 in the TIFF from heterozygous mice was significantly (p < 0.01) elevated, 1.8 fold, compared to normal and homozygous affected animals; normal and homozygous affected animals, however, were not significantly different from each other. The TIFF isolated from homozygous affected mice revealed a 15 fold increase in unesterified cholesterol compared to the TIFF isolated from heterozygous and normal mice. In summary, these findings demonstrate an altered expression of caveolin-1 in liver from heterozygous and homozygous NPC mice and increased concentration of cholesterol from TIFF in homozygous affected NPC mice. The identification of these alterations in the TIFF suggests involvement of detergent insoluble membrane structures, possibly caveolae and/or detergent insoluble glycosphingolipid-enriched complexes (DIGs), in the cholesterol trafficking defect in this disorder.
Subject(s)
Caveolins , Cholesterol/metabolism , Liver/metabolism , Membrane Proteins/genetics , Niemann-Pick Diseases/metabolism , Animals , Biological Transport , Caveolin 1 , Immunohistochemistry , Mice , Mice, Inbred BALB CABSTRACT
Interaction of HDL with cells activates protein kinase C (PKC), a process that may be important in stimulating efflux of excess cellular cholesterol. Here we report that HDL treatment of cholesterol-loaded fibroblasts increases 32P labeling of three acidic phosphoproteins. These phosphoproteins, called pp80, pp27, and pp18 based on apparent M(r) in kD, were also phosphorylated by acute treatment of cells with phorbol myristate acetate, suggesting that they are regulated in response to PKC activation. The HDL-stimulated phosphorylation of pp80 and pp18 was significant after only 30 seconds and was sustained for at least 30 and 120 minutes, respectively, while increased phosphorylation of pp27 was transient, reaching a maximum at 10 minutes. Both pp27 and pp18 were phosphorylated on serine/threonine residues, whereas pp80 was phosphorylated on serine/threonine and tyrosine residues. Immunoprecipitation studies suggested that pp80 is the myristoylated alanine-rich C kinase substrate protein, but the identities of pp27 and pp18 are unknown. HDL and trypsin-digested HDL stimulated phosphorylation of pp80 and pp27, while purified apoA-I, apoA-II, or apoE had no stimulatory effects, indicating that the active component in HDL was trypsin resistant and unlikely to be an apolipoprotein. Conversely, HDL, apoA-I, apoA-II, and apoE all stimulated pp18 phosphorylation, while trypsin-digested HDL had less effect, consistent with pp18's being responsive to HDL apolipoproteins. Treatment of cholesterol-depleted cells with apoA-I also stimulated phosphorylation of pp18, but only transiently. These results suggest that HDL interaction with cells activates diverse PKC-mediated pathways that target different phosphoproteins. Of these three phosphoproteins, only pp18 has a phosphorylation response consistent with its being involved in apolipoprotein-mediated lipid transport.
Subject(s)
Carrier Proteins , Cholesterol/metabolism , Fibroblasts/drug effects , Intracellular Signaling Peptides and Proteins , Lipoproteins, HDL/pharmacology , Membrane Proteins/metabolism , Phosphoproteins/metabolism , Protein Processing, Post-Translational/drug effects , RNA-Binding Proteins , Receptors, Lipoprotein/drug effects , Apolipoprotein A-I/pharmacology , Apolipoprotein A-II/pharmacology , Apolipoproteins E/pharmacology , Biological Transport , Cells, Cultured , Fibroblasts/metabolism , Humans , Male , Myristoylated Alanine-Rich C Kinase Substrate , Phosphorylation/drug effects , Phosphoserine/analysis , Phosphothreonine/analysis , Protein Serine-Threonine Kinases/metabolism , Proteins/metabolism , Receptors, Lipoprotein/physiology , Signal Transduction/drug effects , Skin/cytologyABSTRACT
Human Niemann-Pick type II fibroblasts, which encompass the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) variants, exhibit altered expression of caveolin-1 protein when examined by immunoblotting using an anti-caveolin-1 monoclonal antibody. Unexpectedly, caveolin-1 in heterozygous fibroblasts was significantly elevated as much as 10-fold compared to caveolin-1 in normal and homozygous affected fibroblasts. Homozygous NPC fibroblasts expressed caveolin-1 levels similar to those in normal fibroblasts, while the expression of caveolin-1 in homozygous NPD fibroblasts was slightly elevated. Northern analysis indicates that normal fibroblasts and NPC heterozygous fibroblasts have similar amounts of caveolin-1 mRNA, while NPC homozygous fibroblasts have significantly less caveolin-1 mRNA. In contrast, heterozygous and homozygous NPD fibroblasts exhibit increased levels of caveolin-1 mRNA. These novel findings suggest that caveolin-1 containing subcellular structures are involved in the pathophysiology of Niemann-Pick type II disease. Furthermore, altered caveolin-1 protein expression may serve as a useful marker for the diagnosis of carriers of NPC or NPD.
Subject(s)
Caveolins , Membrane Proteins/biosynthesis , Niemann-Pick Diseases/metabolism , Caveolin 1 , Cells, Cultured , Fibroblasts/metabolism , Heterozygote , Humans , Membrane Proteins/genetics , Niemann-Pick Diseases/geneticsABSTRACT
Acyl-CoA:alcohol transacylase catalyzes the final step in the biosynthesis of storage liquid wax esters from acyl-CoA fatty acids and fatty alcohols in a limited number of microbes, algae, and Simmondsia chinensis Link (jojoba). An improved and automated method of enzyme assay for this catalyst from cotyledons of jojoba is described. The assay method uses reversed-phase C18 high performance liquid chromatography (HPLC) to separate the labeled C30:1 liquid wax product, [14C]-dodecanyl-octadecenoate, from the unreacted substrate, [14C]octadecenoyl-CoA (oleyl-CoA), and other components produced from enzymes present in the crude homogenate of jojoba cotyledons, including [14C]-octadecenoic acid (oleic acid) and [14C]octadecenol (oleyol). Methods are also described for microscale chemical synthesis in one vessel of 14C-radiolabeled substrates and products for the transacylase. These labeled reagents are required to confirm the HPLC separations of reaction products. The radioactive components are quantitated using an on-line flow-through scintillation detector enabling sensitive and precise analysis of the reaction products.