Feasibility of video observed therapy to support controller inhaler use among children in West Baltimore.

OBJECTIVE: To assess feasibility of a novel video directly observed therapy (DOT)-based digital asthma program intended to support correct inhaled corticosteroid (ICS) use among children. METHODS: We conducted a 60-day pilot study among patients 2-18 years attending a primary care clinic with prescribed ICS and sub-optimally controlled asthma (recent hospitalization, ICS nonadherence, frequent rescue inhaler use, therapy escalation, or Asthma Control Test <20). Participants used a mobile application to receive reminders, submit videos of ICS doses (video DOT), and receive asynchronous feedback on adherence and inhaler technique. We assessed enrollment, engagement, program metrics, and user experience; adherence and inhaler errors were secondary outcomes. RESULTS: Of 26 eligible patients, 21 (81%) enrolled and submitted ≥1 video; median age was 11 years (8-15), 71% were male, 90% had Medicaid, and 62% experienced ≥1 exacerbation in the previous 6 months. Retention was 57% and 52% at week 5 and 8, respectively. Participants submitted 810 videos. Missed doses, inhaler errors (n = 247) and adherence issues (n = 107) prompted 543 communications; inadequate inspiration or holding breath were most common. Among 16 patients with engagement >7 days and >4 videos, median inhaler error rate (proportion of videos with ≥1 error) decreased from week 1 to week 2 (73% vs 8%, p ≤ 0.05) with median adherence >80%. Participants experienced the program as long, but easy to use; benefits included building routines, skill, and independence. CONCLUSIONS: This pilot study suggests high program acceptability among our cohort. High engagement with improved inhaler technique over the first 14 days suggests shorter implementation.Supplemental data for this article is available online at at.

Inhibition of intestinal tumor formation by deletion of the DNA methyltransferase 3a.

Aberrant de novo methylation of DNA is considered an important mediator of tumorigenesis. To investigate the role of de novo DNA methyltransferase 3a (Dnmt3a) in intestinal tumor development, we analyzed the expression of Dnmt3a in murine colon crypts, murine colon adenomas and human colorectal cancer using RNA fluorescence in situ hybridization (FISH), quantitative PCR and immunostaining. Following conditional deletion of Dnmt3a in the colon of APC((Min/+)) mice, we analyzed tumor numbers, genotype of macroadenomas and laser dissected microadenomas, global and regional DNA methylation and gene expression. Our results showed increased Dnmt3a expression in colon adenomas of APC((Min/+)) mice and human colorectal cancer samples when compared with control tissue. Interestingly, in tumor tissue, RNA FISH analysis showed highest Dnmt3a expression in Lgr5-positive stem/progenitor cells. Deletion of Dnmt3a in APC((Min/+)) mice reduced colon tumor numbers by ~40%. Remaining adenomas and microadenomas almost exclusively contained the non-recombined Dnmt3a allele; no tumors composed of the inactivated Dnmt3a allele were detected. DNA methylation was reduced at the Oct4, Nanog, Tff2 and Cdkn1c promoters and expression of the tumor-suppressor genes Tff2 and Cdkn1c was increased. In conclusion, our results show that Dnmt3a is predominantly expressed in the stem/progenitor cell compartment of tumors and that deletion of Dnmt3a inhibits the earliest stages of intestinal tumor development.

Management of esophageal perforation after pneumatic dilation for achalasia.

Current management of esophageal perforation after pneumatic dilation for achalasia is thoracotomy and repair with myotomy. This study aims to assess the outcome of patients managed by laparotomy, and the role of laparoscopic repair. The study was carried out by means of retrospective case review and prospective follow-up with a symptom questionnaire. Results were compared with results in patients undergoing elective Heller myotomy. Over a 20-year period, 445 dilations for achalasia were performed in 371 patients. There were 10 esophageal perforations. Nine patients were referred for surgery and were successfully managed with a transabdominal repair. Laparoscopic repair was attempted in four patients but was successful in only one because of the perforation site. After a mean follow-up of 5.4 years, grade 1 or 2 Visick scores were recorded in all patients. Residual symptoms of dysphagia occurred in 67% in the emergency group and 88% in the elective group. There was an increased incidence of heart-burn compared to elective myotomy. Early operation after perforation provides good results for treatment of achalasia. Mild dysphagia persists and there is an increasing sensation of heartburn. The site of perforation is typically posterolateral, which makes laparoscopic repair difficult.

The Ras antagonist, farnesylthiosalicylic acid (FTS), inhibits experimentally-induced liver cirrhosis in rats.

BACKGROUND/AIMS: Protooncogenes may play an important role, not only in carcinogenesis, but also in the regulation of normal cellular proliferation and differentiation. Several studies have indicated increased expression of the Ras protooncogenes in the liver in animal models and in patients with liver cirrhosis. The aim of the present study was to examine whether a synthetic Ras antagonist, S-farnesylthiosalicylic acid (FTS), which specifically dislodges Ras from the membrane of Ras-transformed fibroblasts (EJ cells), can prevent experimentally-induced liver cirrhosis in rats. METHODS: Cirrhosis was induced in male Wistar rats by intraperitoneal administration of thioacetamide (200 mg/kg twice weekly for 12 weeks). The Ras antagonist, farnesylthiosalicylic acid (FTS, 5 mg/kg), was administered during the study period 3 times a week. Ras expression in the liver was determined by Western blot analysis with pan anti-Ras antibodies and by immunohistochemistry. RESULTS: Rats treated with thioacetamide and the Ras antagonist, farnesylthiosalicylic acid (FTS), for 12 weeks had lower histopathologic scores of fibrosis and inflammation (p-values of 0.003 and 0.008, respectively) than those treated with thioacetamide only. There were no differences between the histopathologic scores in vehicle (control) and in Ras-antagonist (FTS) only treatments. Analysis of hepatic hydroxyproline levels from the two thioacetamide-treated groups and controls confirmed the histopathologic scores (7.7+/-0.9 mg/g protein in the TAA-treated vs. 3.8+/-0.5 mg/g protein in the TAA+FTS treated group, p = 0.007). Ras levels, determined by Western blot analysis, were markedly increased in the livers treated with TAA (17-fold over control) and significantly decreased (by about 70%) in the livers of rats treated with TAA and FTS. Studies in isolated human hepatic stellate cells demonstrated that FTS inhibited both DNA synthesis and migration of those cells (p<0.05). CONCLUSION: These results indicate that inhibition of Ras expression in the liver during fibrogenesis, prevents the development of experimentally-induced hepatic cirrhosis.

Expression of matrix metalloproteinase-9 in squamous cell carcinoma of the uterine cervix-clinicopathologic study using immunohistochemistry and mRNA in situ hybridization.

OBJECTIVE: Invasion of the extracellular matrix and blood vessels by malignant neoplasms, with subsequent distant dissemination, is a key event in tumor progression. This process appears to be mediated largely through the action of matrix metalloproteinases (MMPs), a family of proteolytic enzymes produced by both stromal and tumor cells. The role of gelatinases (MMP-2 and MMP-9) in basement membrane and matrix degradation was described in various tumors. We studied MMP-9 protein expression in cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma using immunohistochemistry and detected MMP-9 mRNA using in situ hybridization. METHODS: Fifty squamous cell carcinomas, 10 cases of CIN II-III, and 10 normal cervices were stained for MMP-9, using a monoclonal antibody. The presence of MMP-9 mRNA was studied using in situ hybridization. Results were correlated with patient survival during a follow-up period of up to 167 months (average, 41 months). RESULTS: Immunohistochemical staining of tumor cells for MMP-9 was noted in 36/50 (72%) carcinomas and 5/10 (50%) CIN lesions, but was uniformly absent from the nonneoplastic epithelium adjacent to tumors and from control cervices. Peritumoral staining of stromal cells was observed in 27/50 (54%) carcinomas, but only in 3/10 (30%) CIN lesions and 1/10 (10%) control cervices. The presence of MMP-9 mRNA was detected in tumor cells in 39 (78%) carcinomas and 8 (80%) CIN lesions, but only in 4 (40%) control cervices. An intense signal for MMP-9 mRNA was observed most frequently in carcinomas. MMP-9 mRNA was detected in stromal cells in the majority of cases. However, an intense signal was observed only in stromal cells around invasive tumors. In survival analysis, age (P = 0.016), grade (P = 0. 016), and stage (P = 0.001) showed independent correlation with poor survival. Neither MMP-9 protein expression nor an intense signal for MMP-9 mRNA was associated with poor survival, although the latter was observed more frequently in neoplastic cells of lethal tumors (8/14 tumors vs 11/36 nonlethal tumors). CONCLUSIONS: MMP-9 mRNA and protein expression are elevated in tumor and stromal cells of both high-grade CIN and invasive squamous cell carcinoma of the uterine cervix. Thus, MMP-9 is possibly an early marker of tumor progression in squamous lesions of the cervix. An intense stromal signal for MMP-9 mRNA characterizes some invasive carcinomas. Expression of MMP-9 in cervical carcinoma cells is present in both lethal and nonlethal tumors, consistent with the key role of this proteolytic enzyme in invasion, and does not appear to predict disease outcome.

Variability of facial photographs for use in treatment planning for orthodontics and orthognathic surgery.

Facial photographs are commonly used preoperatively for recording and analyzing skeletofacial and dentofacial deformities prior to orthodontics and/or orthognathic surgery. This study was undertaken to determine the reproducibility of facial photographs over time in any individual patient. Twenty subjects had full-facial frontal and lateral, frontal smile, and lip-to-tooth length (incisor show at rest) photographs taken on 5 different days over a 7- to 14-day period. A total of 18 linear and angular measurements were made on each set of photographs. Standard errors of the 18 measurements over time were generally moderate but varied widely, suggesting that facial photographs have the potential to provide reliable diagnostic information in some cases. In any individual patient, however, there were some variations within measurements that were clinically unacceptable. Measurements made from pictures of the smile were significantly less reliable than those taken at rest, and measurements of vertical head posture and lower lip length were found to be the least reliable. This study suggests that some measurements from facial photographs are generally reproducible over time, but significant individual variations do occur.

Mitochondrial carnitine palmitoyltransferase I isoform switching in the developing rat heart.

The expression pattern of mitochondrial carnitine palmitoyltransferase (CPT) enzymes was examined in the developing rat heart. Whereas the specific activity of CPT II increased approximately 3-fold during the first month of life, the profile for CPT I, which is composed of both liver (L) and muscle (M) isoforms, was more complex. Exposure of mitochondria to [3H]etomoxir (a covalent ligand for CPT I), followed by fluorographic analysis of the membrane proteins, established that while in the adult heart L-CPT I represents a very minor constituent, its contribution is much greater in the newborn animal. Use of the related inhibitor, 2-[6-(2,4-dinitrophenoxy)hexyl]oxirane-2-carboxylic acid (specific for L-CPT I), allowed the activities of the two CPT I variants to be quantified separately. The results showed that in the neonatal heart, L-CPT I contributes approximately 25% to total CPT I activity (in Vmax terms), the value falling during growth of the pups (with concomitant increasing expression of the M isoform) to its adult level of 2-3%. Because the myocardial carnitine content is very low at birth and rises dramatically over the next several weeks, it can be estimated that L-CPT I (Km for carnitine of only 30 microM compared with a value of 500 microM for M-CPT I) is responsible for some 60% of total cardiac fatty acid oxidation in the newborn rat; the value falls to approximately 4% in adult animals. Should these findings have a parallel in humans, they could have important implications for understanding the pathophysiological consequences of inherited L-CPT I deficiency syndromes.

Use of a selective inhibitor of liver carnitine palmitoyltransferase I (CPT I) allows quantification of its contribution to total CPT I activity in rat heart. Evidence that the dominant cardiac CPT I isoform is identical to the skeletal muscle enzyme.

It has recently been established that rat heart mitochondria contain two isoforms of carnitine palmitoyltransferase I (CPT I), the minor 88-kDa variant being identical to liver CPT I (L-CPT I) and the dominant 82-kDa form resembling the skeletal muscle enzyme (M-CPT I) (Weis, B. C., Esser, V., Foster, D. W., and McGarry, J. D. (1994) J. Biol. Chem. 269, 18712-18715). To quantify the functional contribution of L-CPT I to overall CPT I activity in heart mitochondria a selective inhibitor of the former was needed. The dinitrophenol analog of 2[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylic acid (etomoxir) (DNP-Et) was found to have this property. When liver and skeletal muscle mitochondria were exposed to DNP-Et in the presence of ATP and CoASH, the DNP-Et-CoA formed completely inhibited liver CPT I while leaving the muscle enzyme unaffected. Similar treatment of heart mitochondria blocked only the L-CPT I component. This had the effect of shifting the apparent Km for carnitine from approximately 200 to approximately 500 microM and the I50 value for malonyl-CoA (the concentration needed to suppress enzyme activity by 50%) from approximately 0.18 to approximately 0.06 microM, i.e. the heart system now behaved exactly the same as that from skeletal muscle. Taking the Km for carnitine of L-CPT I and M-CPT I to be 30 and 500 microM, respectively, it could be calculated that the former contributes approximately 2% to the total CPT I in heart. When the 82-kDa CPT I isoforms of heart and skeletal muscle were labeled with [3H]etomoxir and then exposed to trypsin, the fragmentation patterns obtained were identical and quite distinct from that given by CPT I from liver. We conclude that (i) DNP-Et, unlike other agents of the oxirane carboxylic acid class, has remarkable inhibitory selectivity for L-CPT I over M-CPT I; (ii) the previously puzzling observation that rat heart CPT I displays kinetic characteristics intermediate between those of the enzymes from liver and skeletal muscle is entirely accounted for by the low level expression of L-CPT I in the cardiac myocyte; and (iii) the dominant 82-kDa CPT I isoform in heart is identical to the muscle enzyme. The data reaffirm that, in contrast to CPT II, CPT I exists in at least two isoforms and that both are present in rat heart.

Rat heart expresses two forms of mitochondrial carnitine palmitoyltransferase I. The minor component is identical to the liver enzyme.

To begin to explore the basis for the tissue-specific expression of mitochondrial carnitine palmitoyltransferase I (CPT I), we focused on three rat tissues (liver, heart, and skeletal muscle) in which the enzyme was known to display very different properties. In Northern blot analysis, a cDNA probe corresponding to liver CPT I readily hybridized to a 4.5-kilobase species of mRNA in liver and heart, but not in skeletal muscle. Using the same probe to screen a neonatal rat heart cDNA library, a full-length clone, surprisingly having 100% sequence identity to the liver CPT I cDNA, was isolated. The paradox was resolved by two additional experiments. First, in Western blots of mitochondrial membranes, an antibody raised against liver CPT I recognized the 88-kDa protein in heart, as well as in liver, but not in skeletal muscle. Second, high specific activity [3H]deschloroetomoxir (a covalent ligand for CPT I) reacted with a single form of CPT I in liver (approximately 88 kDa) and skeletal muscle (approximately 82 kDa), while proteins of both sizes were labeled in the cardiac myocyte. Tritiated ligand binding to the two heart proteins was blocked by excess unlabeled malonyl-CoA. It is concluded that liver and skeletal muscle each contains a single and distinct isoform of CPT I with monomeric size of approximately 88 and 82 kDa, respectively. The heart contains a CPT I protein of approximately 82 kDa in size (probably identical to the skeletal muscle protein) but, importantly, also expresses the liver-type enzyme. The results likely explain why previous studies of heart CPT I yielded an apparent Km for carnitine and I50 value for malonyl-CoA inhibition that were intermediate between those of the liver and skeletal muscle enzymes.

Cloning, sequencing, and expression of a cDNA encoding rat liver carnitine palmitoyltransferase I. Direct evidence that a single polypeptide is involved in inhibitor interaction and catalytic function.

We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase I (CPT I). Oligonucleotides corresponding to two tryptic peptides derived from the malonyl-CoA/etomoxir-CoA-binding protein of rat liver mitochondria (Esser, V., Kuwajima, M., Britton, C. H., Krishnan, K., Foster, D. W., and McGarry, J. D. (1993) J. Biol. Chem. 268, 5810-5816) were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone obtained consisted of a 102-nucleotide 5'-untranslated region, a single open reading frame of 2,319 bases predicting a protein of 773 amino acids (M(r) = 88,150), and a 3'-untranslated segment of 1,957 nucleotides followed by the poly(A)+ tail. A 0.9-kilobase fragment of the cDNA recognized a single species of mRNA (approximately 4.7 kilobases in size) in rat liver. The identity of the cDNA was confirmed by the findings that (i) the open reading frame encoded all four peptides found in the original protein; (ii) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV6, resulted in a selective and 10-20-fold induction of a malonyl-CoA- and etomoxir-CoA-sensitive CPT activity; and (iii) the overexpressed product was readily detected on Western blots by an antibody raised against the starting material. It seems likely that the de novo synthesized enzyme is targeted to the mitochondrial outer membrane via a leader peptide and that the mature protein achieves membrane anchoring through a stretch of 20 amino acids present near its amino terminus. The predicted amino acid sequence of the protein shows regions of strong identity with those of three other rat acyltransferases, namely, liver CPT II, liver carnitine octanoyltransferase, and brain choline acetyltransferase. The findings provide the first insight into the structure of a CPT I isoform. They also establish unequivocally that CPT I and CPT II are distinct proteins and that inhibitors of CPT I interact within its catalytic domain, not with an associated regulatory component.

Phosphatidyl-Tris rather than N-acylphosphatidylserine is synthesized by Rhodopseudomonas sphaeroides grown in Tris-containing media.

We have synthesized 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(N-oleoyl)serine (N-acyl-PS) and 1,2-dioleoyl-sn-glycero-3-phospho-Tris (phosphatidyl-Tris) and have characterized both phospholipids by their chemical and chromatographic properties, as well as by their IR, 13C NMR, and 1H NMR spectra. Comparison of these data with those reported for a phospholipid isolated from Rhodopseudomonas sphaeroides grown in Tris-supplemented media [Donohue et al. (1982) Biochemistry 21, 2765-2773] indicates that R. sphaeroides synthesizes phosphatidyl-Tris rather than N-acyl-PS.

New insights into the mitochondrial carnitine palmitoyltransferase enzyme system.

Dissection of the mitochondrial carnitine palmitoyltransferase (CPT) enzyme system in terms of its structure/function relationships has proved to be a formidable task. Although no one formulation has gained universal agreement we believe that the weight of evidence supports a model with the following features: a) in any given tissue CPT I and CPT II are distinct proteins; b) CPT I, unlike CPT II, is detergent labile; c) within a species CPT II is expressed body wide, whereas CPT I exists as tissue specific isoforms; d) malonyl-CoA and other CPT I inhibitors probably interact at the catalytic center of the enzyme, not with a regulatory subunit. The amino acid sequences of rat and human CPT II (deduced from cDNA clones) show them to be similar proteins (greater than 80% identity) but encoded by mRNAs of significantly different sizes. Efforts to clone and sequence the cDNA for rat liver CPT I are presently underway.

Peroxidative modification of phospholipids in myocardial membranes.

Rat heart myocardial membranes exposed to the free radical generating system, Fe2+/ascorbate, undergo lipid peroxidation as evidenced by the accumulation of thiobarbituric acid-reactive substances, loss of polyunsaturated fatty acids from phospholipids, and formation of conjugated dienes and fluorescent substances. In addition, the treated membranes exhibit a dramatic decrease in extractable phospholipids. This decrease is even more pronounced in individual phospholipid classes isolated by high-performance liquid chromatography. The decrease in lipid phosphorus under oxidant stress is accompanied by an increase in the phosphorus content of the aqueous phase after Folch extraction and by an even greater increase of phosphorus in the protein residue. In addition, increased amounts of saturated and monounsaturated fatty acyl groups are found in the protein residue of Fe2+/ascorbate-treated membranes. Extraction of the oxidant-treated membranes with acidic solvents does not enhance the recovery of phospholipids and neither does treatment with detergents, trypsin, and chymotrypsin prior to lipid extraction. However, treatment with the bacterial protease, Pronase, markedly enhances the recovery of phospholipids from the peroxidized membranes. These results indicate that membrane phospholipids undergoing free radical-induced peroxidation may form lipid-protein adducts, which renders them inextractable with lipid solvents.

Cloning, sequencing, and expression of a cDNA encoding rat liver mitochondrial carnitine palmitoyltransferase II.

We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase II (CPT II). Beginning with the purified protein CNBr fragments were generated and sequenced. Corresponding oligonucleotides were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone ultimately obtained consisted of a 62 nucleotide 5'-untranslated region, a single open reading frame of 1,974 bases predicting a protein of 658 amino acids (Mr = 74,119), and a 3'-untranslated segment of 260 nucleotides followed by the poly (A) tail. The identity of the cDNA was confirmed by the findings that (a) the open reading frame encoded all three peptides found in the original protein; (b) a fourth peptide synthesized from a portion of the deduced amino acid sequence and used to immunize a rabbit resulted in the generation of an antibody that recognized pure CPT II on a Western blot; (c) in vitro transcription and translation of the cDNA (ligated into pBlue-script KS (+] generated a protein that was specifically immunoprecipitated by anti-CPT II antibody and having a Mr slightly greater than that of mature CPT II; (d) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV4, resulted in a 6-fold induction of mitochondrial CPT II catalytic activity. It seems likely that the de novo synthesized enzyme gains entry into the mitochondrion via a targeting peptide that is subsequently cleaved. The mature protein probably associates (relatively loosely) with the inner membrane through a limited number of membrane spanning domains. The predicted amino acid sequence of CPT II shows strong identity with those of two other acyltransferases, namely, rat liver peroxisomal carnitine octanoyltransferase and porcine choline acetyltransferase.

Inter-tissue and inter-species characteristics of the mitochondrial carnitine palmitoyltransferase enzyme system.

Properties of the carnitine palmitoyltransferase (EC 2.3.1.21) (CPT) enzyme system were compared in isolated mitochondria from a range of tissues in rodents, monkey, and man. Common features were as follows: (a) while membrane-bound, CPT I, but not CPT II, was inhibited reversibly by malonyl-coenzyme A (CoA) and irreversibly by CoA esters of certain oxirane carboxylic acids; (b) the detergent, Tween-20, readily solubilized CPT II in active form while leaving CPT I membrane associated and catalytically functional; (c) octyl glucoside and Triton X-100 released active CPT II but caused essentially complete loss of CPT I activity. Use of [3H]tetradecylglycidyl-CoA, a covalent ligand for CPT I, yielded estimates of the enzyme's monomeric molecular size: approximately 86 kDa in non-hepatic tissues and approximately 90-94 kDa in liver, depending upon species. A polyclonal antibody to purified rat liver CPT II recognized a single protein in each tissue; its apparent molecular mass was approximately 70 kDa in all rat tissues and approximately 68 kDa in all mouse tissues as well as monkey and human liver. On Northern blot analysis a rat liver CPT II cDNA probe detected a single approximately 2.5-kilobase mRNA in all rat and mouse tissues examined. The following points are emphasized. First, CPT I and II are different proteins. Second, within a species CPT II, but not CPT I, is probably conserved across tissue lines. Third, slight variations in size of both enzymes were found in different species, although, at least in the case of CPT II, significant amino acid identity exists among the various isoforms. Fourth, CPT I, unlike CPT II, requires membrane integrity for catalytic function. Finally, the strategic use of detergents provides a simple means of discriminating between the two enzyme activities.

Assay of cardiolipin peroxidation by high-performance liquid chromatography.

Commercial preparations of bovine cardiolipin (diphosphatidylglycerol) in chloroform solution contain substantial amounts of oxidation products. These oxidized derivatives, characterized by the presence of varying amounts of hydroperoxides and conjugated dienes, can be separated from unoxidized cardiolipin by normal phase high-performance liquid chromatography (HPLC) using UV detection. When purified cardiolipin is subjected to autoxidation in aqueous media, oxidation products of similar HPLC properties are produced. Storage of cardiolipin in chloroform induces both autoxidation and hydrolysis whereas storage in ethanol and other solvents does not. It is recommended not to use chloroform for the long-term storage of cardiolipin.

Hydrolysis of N-acylated glycerophospholipids by phospholipases A2 and D: a method of identification and analysis.

We have previously identified N-acylethanolamine phospholipids in infarcted dog heart and in normal fish brain by chemical and enzymatic degradation. We now report that hydrolysis with phospholipase D from Streptomyces chromofuscus removes N-acylethanolamine from N-acylethanolamine phospholipids and lyso N-acylethanolamine phospholipids, or N-acylserine from lyso N-acylserine phospholipids. At acidic pH, a phosphatase present in the phospholipase D preparation further hydrolyzes the resulting phosphatidic acid (PA) or lyso-PA to diacyl- or monoacylglycerol. Because N-acylserine phospholipids are a poor substrate for the phospholipase D, pretreatment with phospholipase A2 (Trimeresurus flavoviridis venom) is used to remove the 2-O-acyl group. Thus, both types of N-acylated phospholipids can be analyzed by consecutive phospholipase A2 and phospholipase D treatment. Reaction products, i.e., free fatty acids, monoacylglycerols and N-acylethanolamine or N-acylserine, are separable by thin-layer chromatography. Both N-acyl components can be further characterized by conversion to the t-butyldimethylsilyl derivatives. The method was used to identify and analyze the N-acylserine phospholipids of bovine brain.