Increased saturated triacylglycerol levels in plasma membranes of human neutrophils stimulated by lipopolysaccharide.

Neutrophils isolated from patients with bacterial infections or stimulated in vitro with lipopolysaccharide (LPS) produce a high resolution, lipid-dominated spectrum on 1H-NMR spectroscopy (May et al, 1993. J. Infect. Dis. 168: 386-392). We have investigated the origin of this lipid signal using NMR and chemical analyses of both whole neutrophils and purified plasma membranes. Plasma membranes from neutrophils that had been stimulated with 50 microg/ml LPS exhibited the high resolution 1H-NMR signal, and contained double the triacylglycerol (TAG) content of plasma membranes isolated from resting cells. Chemical analysis of the whole cells indicated that the TAG also increased at the cellular level (1.7-fold) after stimulation with LPS. Diradylglycerol increased 2- to 3-fold in both whole cells and plasma membranes after stimulation, but was only a minor component compared with TAG. The plasma membrane protein/phospholipid ratio increased 2.6-fold, whereas cholesterol (free and esterified) was unchanged. The membranes from LPS-stimulated neutrophils exhibited increased fluidity, as judged by increased merocyanine 540 binding, consistent with a 2-fold reduction in cholesterol/phospholipid ratio. LPS induced a shift in fatty acid content of whole cell polar lipids towards more oleic acid and less palmitic acid, whereas the neutral lipid fraction contained increased amounts of palmitic and stearic acids. The TAG fraction of plasma membrane lipids contained increased amounts of palmitic acid when prepared from cells stimulated with LPS. We conclude that the 1H-NMR signal in LPS-stimulated neutrophils arises from increased amounts of plasma membrane TAG with an elevated content of palmitic acid.

Changes in cellular and plasma membrane phospholipid composition after lipopolysaccharide stimulation of human neutrophils, studied by 31P NMR.

Lipopolysaccharide (endotoxin, LPS) exerts potent proinflammatory effects on neutrophils which may involve membrane phospholipid metabolism. The cellular and plasma membrane phospholipid composition of resting neutrophils and those stimulated with 50 microg ml(-1) LPS were studied by 31P NMR and chemical analysis. A rapid new method for plasma membrane purification was employed, involving the direct lysis of cytoplasts. Chemical analyses showed that, although total cellular phospholipid content did not change with LPS stimulation, there was twice the amount of phospholipid present in plasma membranes isolated from stimulated cells, resulting in a lowered cholesterol/phospholipid ratio. Since internal membranes have lower cholesterol content this result is consistent with an origin from insertion of these membranes (most probably from the endoplasmic reticulum) into the plasma membrane, thereby increasing its fluidity. The individual phospholipid classes of both cells and membranes were quantified by 31P-NMR spectroscopy after dissolution in sodium cholate without prior extraction of lipids, allowing partial resolution of the major phospholipid classes and ether-linked phospholipids. Ether-linked lipids were distinguished from diacyl phospholipids by hydrolysis of lipid extracts with HCl and phospholipase A1, There was a significant increase in phosphatidylserine in both cells and plasma membranes after stimulation, with a decrease in the phosphatidylethanolamine (diacyl and plasmalogen) content in the cells. Plasma membranes from stimulated cells exhibited a significant decrease in a phospholipid tentatively identified as 2-arachidonoyl-1-alkyl-sn-glycero-3-phosphocholine, a precursor of the lipid inflammatory mediator, platelet-activating factor. This report is the first to elaborate the changes in phospholipid composition in human neutrophils as a whole, and in plasma membranes separated from them, before and after stimulation by the physiological activator, LPS.

Two-dimensional proton magnetic resonance spectroscopy for tissue characterization of thyroid neoplasms.

We have previously demonstrated that one dimensional (1D) proton (1H) magnetic resonance spectroscopy (MRS) can distinguish normal thyroid tissue from thyroid carcinoma using a spectral ratio of peak intensity at 1.7 ppm/0.9 ppm. Two dimensional (2D) 1H-MRS allows identification of specific molecules that have overlapping peaks in the 1D-MR spectrum. Specimens from 93 consecutive thyroid nodules were examined using 2D 1H-MRS on a Bruker AM-360 wide-bore spectrometer. There was a progressive increase in lipid cross peaks assigned to di-/triglycerides when comparing colloid/hyperplastic nodules to follicular adenoma, and adenoma to carcinoma. A specific cross peak attributable to cholesterol/cholesteryl esters was commonly seen in carcinomas. In contrast, two unassigned cross peaks unique to the thyroid were more prevalent in benign lesions. There was an overall increase in cross peaks attributable to cell surface fucosylation in carcinoma when compared to benign lesions, although the fucose spectral pattern was not specific for cancer. On this basis, a spectral ratio of peak intensity at 2.05 ppm/0.9 ppm more clearly distinguished benign follicular adenoma from carcinoma. 2D 1H-MRS thus identifies chemical changes that allow more specific tissue characterization of thyroid neoplasms.

Characterization of human ovarian epithelial tumors (ex vivo) by proton magnetic resonance spectroscopy.

Proton magnetic resonance spectroscopy (1H MRS) offers an alternative investigational modality that will assist current pathologic techniques in the diagnosis of human ovarian epithelial tumors. Histologically normal human ovarian tissue (n = 12) was compared with ovarian benign fibromas (n = 3) and surface epithelial-stromal tumors (benign, n = 18; proliferating, n = 9; frankly malignant, n = 30) ex vivo by 1H MRS. The distinction between carcinomatous and benign or normal tissue (P<0.0001; Student's t-test) was made on one-dimensional (1-D) 1H MR spectra utilizing differences in resonance intensities of cellular lipid, creatine/phosphocreatine and lysine. The sensitivity and specificity of the test were 87% and 91%, respectively. Two-dimensional (2-D) MRS of carcinomatous biopsies showed multiple crosspeaks attributable to cell-surface fucosylation that correlated with tumor grade and loss of cellular differentiation. The multiple fucose crosspeaks were absent in spectra from normal ovary and benign tumors. The distinction between carcinomatous and normal or benign tissue based on MR-visible fucosylation gave a sensitivity and specificity of 88% and 97%, respectively. Proliferating tumors exhibited a range of cell-surface fucosylation patterns indicative of malignant potential.

Diagnosis of follicular thyroid lesions by proton magnetic resonance on fine needle biopsy.

Most thyroidectomies are currently performed for diagnostic purposes. It has been established that proton magnetic resonance spectroscopy (MRS) on excised thyroid tissue can distinguish normal thyroid from invasive carcinomas (P < 0.0001). The purpose of this study was to assess whether the same discrimination could be obtained preoperatively from fine needle biopsy (FNB). This has clinical importance because cytological examination of fine needle aspirates cannot distinguish between benign and malignant follicular thyroid lesions. Here we demonstrate a sensitivity of 95% for proton MRS to correctly identify clinically or histologically proven carcinoma. MRS measurements were made on FNB specimens (containing as few as 10(6) cells) from solitary thyroid nodules. MR assessment of FNB was inconsistent with that of the corresponding tissue in only 6.5% of cases. The discrimination between cancer and normal tissue was based on altered cellular chemistry measured as a one-dimensional spectral ratio of resonances from the amino acid lysine and lipid. Benign follicular lesions were separated into two groups: 67% with a spectral ratio similar to malignant thyroid tumors, and 33% with a spectral ratio comparable to that in normal thyroid tissue. Thus, in contrast with histopathology, MRS offers a method for assessment of FNB of follicular lesions with the potential to identify a biologically benign group, which could avoid thyroid surgery for purely diagnostic purposes.

Proton magnetic resonance and human thyroid neoplasia. II: Potential avoidance of surgery for benign follicular neoplasms.

Thyroid cancer is rare, but many thyroidectomies continue to be performed simply to exclude a diagnosis of malignancy. The purpose of this study was to determine the potential financial savings associated with the use of proton magnetic resonance analysis of follicular neoplasms. Proton magnetic resonance spectroscopy was performed on tissue obtained at the time of surgery from 98 consecutive solitary or dominant thyroid nodules. Fine-needle biopsies were also performed on operative specimens, and the tissues assessed by proton magnetic resonance; these spectra were then compared with those obtained from tissue specimens. An estimate of potential savings was obtained by comparing the magnetic resonance data with the indications for surgery and pathology on all patients having thyroidectomy over a 10-year period. Proton magnetic resonance spectroscopy was able to distinguish between normal thyroid tissue and invasive thyroid cancer with 100% specificity. Benign follicular adenomas fall into two groups: 44% having a spectral pattern comparable with normal thyroid, and the remaining 56% demonstrating an altered spectral pattern more comparable to the malignant magnetic resonance profile. Proton magnetic resonance spectroscopy on fine-needle biopsy specimens produced spectra similar to those from tissues from the same patient. From a fine-needle biopsy specimen, proton magnetic resonance spectroscopy can identify a group of benign follicular adenomas with spectral profiles akin to those of normal thyroid cells, thus avoiding the need for unnecessary surgical excision. The potential savings in one surgical unit alone were over $1 million in 10 years.

Circulating polymorphonuclear leukocytes from patients with gram-negative bacteremia are not primed for enhanced production of leukotriene B4 or 5-hydroxyeicosatetraenoic acid.

The hypothesis was tested that polymorphonuclear leukocytes (PMNL) from patients with gram-negative bacteremia are primed to produce leukotriene B4 (LTB4) or 5-hydroxyeicosatetraenoic acid (5-HETE), in response to concentrations of calcium ionophore A23187, which are substimulatory for control PMNL. PMNL from 11 bacteremic patients and 8 healthy subjects (11 samples) produced similar quantities of LTB4, omega-oxidation products of LTB4, and 5-HETE after incubation with 0.3 and 0.5 microM A23187 for 5 min. At the detection threshold of 0.3 microM A23187, LTB4 was present in PMNL preparations from 9 of 11 patients and 7 of 11 control samples and 5-HETE from the same 9 patients and from 6 controls. There was no correlation between LTB4 or 5-HETE and plasma levels of endotoxin. In this group of patients, priming of PMNL by gram-negative bacteremia did not lead to enhanced production of LTB4, its omega-oxidation products, or 5-HETE when PMNL were challenged with low concentrations of A23187.

Proton magnetic resonance and human thyroid neoplasia. I: Discrimination between benign and malignant neoplasms.

PURPOSE: Thyroid nodules are very common, yet the vast majority are biologically benign. The extreme difficulty facing the clinician selecting potentially malignant thyroid nodules for surgery was the subject of a recent editorial by Ernest L. Mazzaferri in the American Journal of Medicine (93:359-362, 1992). Here we evaluate the potential of proton magnetic resonance spectroscopy (1H MRS) to provide a solution to this problem. PATIENTS: Thyroid tissue from fifty-three patients undergoing partial or total thyroidectomy for solitary thyroid nodules were assessed by 1H MRS. RESULTS: When compared with the histologic diagnosis, 1H MRS distinguished normal thyroid tissue (n = 8) from invasive papillary (n = 9), anaplastic (n = 1), and medullary (n = 1) carcinomas with P values of < 0.0001, based on altered cellular chemistry. The same magnetic resonance (MR) criteria categorized pathologically proven follicular carcinoma (n = 8) (established as such by the presence of capsular or vascular invasion at the periphery of the tumor, or by the presence of metastases in the patient) with the other thyroid cancers (P < 0.0001). All other "benign" follicular neoplasms (n = 34), including five atypical follicular adenomas, were assessed by the same 1H MRS criteria and found to fit into one of the two above categories, viz. analogous to benign or malignant thyroid tissue. CONCLUSIONS: Proton MRS has the potential to separate out a group of truly benign follicular neoplasms from follicular tumors (both follicular adenomas and follicular carcinomas) that have an atypical follicular pattern on cytologic examination. This is the first report of an objective diagnostic procedure that has the potential to obviate surgical excision in a significant number of patients with benign follicular adenomas, independent of exhaustive histopathologic assessment.

The presence of cytoplasmic lipid droplets is not sufficient to account for neutral lipid signals in the 1H MR spectra of neutrophils.

Stimulation of human peripheral blood neutrophils with lipopolysaccharide (LPS), arachidonic acid (AA) and oleic acid (OA) resulted in significant increases in cytoplasmic lipid droplets. This phenomenon was also observed in enucleated and degranulated cytoplasts prepared from neutrophils stimulated with LPS. In contrast, only LPS and high concentrations of OA (10 microM) produced an increase in the lipid intensities of the MR spectra of neutrophils as determined by COSY cross peak volume measurements. Lipid intensities in cells stimulated with OA (2.5 microM) and AA (2.5 microM) and phorbol myristate acetate (20 nM) were not elevated. LPS stimulation of resting cytoplasts resulted in increased lipid droplets but not MR lipid intensities. These data suggest that while cytoplasmic lipid droplets may correlate with MR lipid intensity under some circumstances, their presence is not sufficient to account for increased neutral lipid signals.

Proton magnetic resonance spectroscopy of polymorphonuclear leukocytes from patients with serious bacterial infections.

Triacylglycerols in human neutrophils exposed to proinflammatory stimuli generate a high-resolution proton magnetic resonance (1H MR) spectrum. Lipid cross-peak F volumes in neutrophils from patients with inflammatory conditions were measured. Values in patients hospitalized with localized infections (14.4 +/- 9.0; mean +/- SD) or bacteremia (19.3 +/- 9.7) were significantly higher than in patients with noninflammatory conditions (6.2 +/- 5.3) and healthy controls (2.0 +/- 3.0; P < .001). The positive predictive value of F volumes > 10 was 93% for all infection; the negative predictive value of volumes < or = 10 was 68% for all infection and 92% for bacteremia. Plasma lipopolysaccharide (LPS) concentrations were highest in bacteremic patients but did not correlate with levels of tumor necrosis factor-alpha (TNF alpha) or interleukin-6. In vitro, LPS increased F volumes of control neutrophils from 2.0 +/- 3.0 to 37.2 +/- 6.7 (P < .001); TNF alpha had no effect. F volumes in 1H MR spectra may be useful clinically to discriminate between serious bacterial infection and other inflammatory conditions. TNF alpha is not the stimulus for generation of lipid spectra in vivo.

Esterified cholesterol and triglyceride are present in plasma membranes of Chinese hamster ovary cells.

The chemical composition of highly purified plasma membrane preparations from a series of malignant Chinese hamster ovary (CHO) cell lines were undertaken to ascertain if neutral lipid, including cholesteryl ester and triacylglycerol, were present. Triacylglycerols (33-41 nmol/mg total lipid) and cholesteryl ester (226-271 nmol/mg) were measured in the plasma membranes and differences in the chemical composition of these membranes recorded. The most significant difference was a gradual decrease in the level of free cholesterol from wild type (312 +/- 7 nmol/mg total plasma membrane lipid), Pod RII-6 (268 +/- 64 nmol/mg total plasma membrane lipid), Col R-22 (243 +/- 39 nmol/mg total plasma membrane lipid) to EOT (204 +/- 20 nmol/mg total plasma membrane lipid), with a concomitant increase in the degree of saturation of the cholesteryl ester fatty acids, particularly palmitic acid. No statistically significant differences were apparent in the chemical composition of the whole cells in this series. The one-dimensional (1D) 1H-NMR spectra of the four malignant cell lines showed a gradation in intensity of lipid resonances, in the order of wild type, Pod RII-6, Col R-22 and EOT, with EOT having the strongest lipid spectrum. Interestingly, the increase in acyl-chain signal intensities in the 1H-NMR spectra of this series of CHO cells and emergence of signals from cholesterol and/or cholesteryl ester, coincide with alterations in the amount of free cholesterol and the degree of saturation of the fatty-acyl chain of the esterified cholesterol in the plasma membranes. It is our hypothesis that, together, cholesteryl ester and triacylglycerol form domains in the plasma membrane and that when the cholesteryl ester has a largely saturated fatty acid content, the lipids are in isotropic liquid phase and hence visible by NMR.

GMP-140 (P-selectin) inhibits human neutrophil activation by lipopolysaccharide: analysis by proton magnetic resonance spectroscopy.

Proton magnetic resonance spectroscopy has been used to monitor the effect of GMP-140 on the stimulation of human peripheral blood neutrophils. Stimulation of neutrophils by lipopolysaccharide gives rise to a high resolution lipid spectrum from the intact cells. Fluid phase GMP-140, which prevents adhesion and development of inflammatory responses of neutrophils, was found to inhibit these changes in the lipid spectrum by up to 40%. Anti-GMP-140 Fab fragments reversed this effect while non-immune Fab fragments did not affect the observed inhibition by GMP-140.

Comparison of human polymorphonuclear leukocytes from peripheral blood and purulent exudates by high resolution 1H MRS.

Human pus samples from various sites, including soft tissue and pleural cavity, as well as sputum from patients with cystic fibrosis have been analyzed by 1H magnetic resonance spectroscopy (MRS) and found to generate high resolution spectra. Assignments have been made for neutral lipid, amino acids, taurine, and lactate. Human peripheral blood polymorphonuclear leukocytes have also been examined and a similar spectrum containing these lipid and metabolite resonances was detected in cells stimulated with lipopolysaccharide. In unstimulated cells, only resonances from taurine and lactate were observed, suggesting that the presence of the lipid and metabolite resonances is an indicator of cellular activation of polymorphonuclear leukocytes. This is the first report of 1H MRS applied to intact human polymorphonuclear leukocytes and extends available documentation as to which types of normal and/or diseased tissue contain MR visible molecules.

Tumour evolution in rats monitored by changes to serum and lipoproteins.

The biochemical, physicochemical and magnetic resonance (MR) properties of rat serum allow tumour evolution to be monitored. Serum from female Fischer rats injected with rat mammary adenocarcinoma cells contained a low-density lipoprotein (LDL)-like lipoprotein and decreased high-density lipoprotein levels compared with normal rat serum. Increases in secondary tumour burden coincided with enlarged LDL-like particles, altered MR properties and elevation of serum triglyceride. By fucosidase treating metastatic R13762 cells prior to injection, not only was metastatic capacity retarded, but serum lipoproteins were also altered. These physicochemical alterations suggest an intricate relationship between both primary and secondary tumour burdens and the serum lipoprotein profile.

Vinblastine sensitivity of leukaemic lymphoblasts modulated by serum lipid.

The high-resolution proton magnetic resonance spectrum of leukaemic lymphoblasts is characteristic of neutral lipid in an isotropic environment. When such lymphoblasts are selected for resistance to the anticancer drug vinblastine, the intensity of this spectrum increases with increasing drug resistance. A reversal of this trend can be achieved by growing cells in delipidated serum, whereby lipid spectrum and drug resistance are diminished. However, both can be restored by subsequent regrowth in normal medium. Thus, although detectable genetic changes accompany the development of vinblastine resistance, the expression of these changes can be modulated by environmental lipid.

Plasma membrane lipid composition of vinblastine sensitive and resistant human leukaemic lymphoblasts.

Plasma membranes purified 32- to 45-fold were isolated from leukaemic T-lymphoblasts, both sensitive and resistant to the Vinca alkaloid vinblastine. On development of drug resistance there was a very significant elevation of ether lipid content. 1-0-alkyl phospholipid increased by 200% with a smaller 30% increase in 1-0-alkenyl phospholipid. Cholesterol and phospholipid levels were also found to increase by 50% and 30% respectively, while the lipid to protein ratio increased by 60%. More modest changes were observed in the fatty acid composition of the membranes, with an alteration in the double bond index from 35.3 to 41.2. These lipid changes may have important implications in the changes to membrane permeability that develop with drug resistance.

Inhibition of metastatic potential by fucosidase: an NMR study identifies a cell surface metastasis marker.

NMR spectroscopy is able to detect subtle changes to the surface chemistry of cells. We have previously shown that high-resolution 1H NMR methods can identify tumor cells with the capacity to metastasize, and we now report that the long T2 relaxation value (500-800 ms) observed in metastatic rat mammary adenocarcinoma cells is removed by treatment with fucosidase. Two-dimensional scalar-correlated NMR (COSY) spectra of fucosidase-treated cells show that a cross peak, consistent with scalar coupling between the methyl and methine groups on fucose and usually associated with malignancy and metastatic ability, is absent. Metastases were observed in only two out of ten rats injected subcutaneously with enzyme-treated cells compared to eight out of ten with untreated cells. NMR studies on isolated cellular lipids identified the long T2 relaxation value only in the ganglioside fraction. This fraction accounts for 51% of the total 14C-labelled fucose incorporated into the cells. We propose that fucogangliosides are an indicator of metastatic potential in rats. The observation that a cell surface metastasis marker has an NMR signal with a characteristically long relaxation value has important consequences for the future use of magnetic resonance imaging and spectroscopy in the cancer clinic.

Hyperlipidemia as a biochemical basis of magnetic resonance plasma test for cancer.

An increase in the plasma levels of apoprotein B-containing lipoproteins is the basis of the magnetic resonance (MR) test for cancer. The narrow MR line width reported by Fossel and co-workers to be associated with the presence of malignant disease is due to a relative increase of very low density lipoprotein. In contrast, the plasma from healthy controls, which has a much broader spectrum, has a higher proportion of high density lipoprotein. However, plasma from patients with hyperlipidemia unrelated to cancer also show narrow MR line widths and are therefore a confounding variable. We used magnetic resonance spectroscopy (MRS) to assess the plasma from 253 patients with a range of lipid related diseases and cancer, and 28 controls. A significant difference (p less than or equal to 0.0005) of 10 Hz exists between the mean line width of the controls and hyperlipidemics without malignant disease. However, in patients with solid tumours a difference of 7 Hz (p less than or equal to 0.0005) in the mean values is recorded although there is an overlap of 6 Hz compared with the controls. Moreover the MRS method was not found to distinguish patients with lymphomas from the control population. The index was not found to be related to patient age or tumour burden.

A comparison of the chemical analyses of cell lipids with their complete proton NMR spectrum.

Whole cells are made up of molecules in different environments to which NMR spectroscopy is sensitive. In particular, malignant and transformed cells contain lipids not only in bilayers but in isotropically tumbling domains which give rise to high-resolution spectra. We have recently developed a technique for simultaneously analyzing broadline and high-resolution signals (M. Bloom, K. T. Holmes, C. E. Mountford, and P. G. Williams, J. Magn. Reson., in press) and we report here its application to a range of rat, mouse, and human cell lines. Some selected features of the NMR spectra were compared with the chemical analysis of the whole-cell lipid. We found that in general the proportion of protons in the narrow methylene resonance at 1.3 ppm increased with the neutral lipid content of the cells. This peak was chosen because its T2 relaxation behavior correlates with metastatic potential in a rat model system. This new technique could be applied to other high-resolution components both in healthy and in diseased states.

Proteolipid identified by magnetic resonance spectroscopy in plasma of a patient with borderline ovarian tumour.

Magnetic resonance spectroscopy (MRS) can identify abnormal lipoproteins in the plasma of patients with premalignant and malignant tumours. Proteolipid complexes, 8-11 nm and 25-28 nm in size, were isolated from the plasma of a patient with a borderline ovarian tumour. These complexes, which generated a characteristically long MRS T2 relaxation value (greater than 400 ms), were disrupted by ribonuclease. None of the conventional lipoproteins had a T2 value above 160 ms. Chemical analysis of the proteolipid complexes showed a 20% glycolipid component, and MRS identified a fucosylated molecule as the origin of the long T2 value. 9 months after resection of all tumour, a visible lipoprotein band, possibly lipoprotein (a), persisted in the plasma but neither the long T2 relaxation value nor the 8-11 nm or 25-28 nm particles were present. The long T2 relaxation value in the MRS profile, found in isolated proteolipid and unfractionated plasma and serum of other patients with carcinoma of the ovary and colon, provides a non-invasive method of assaying for cancer.