Quantitative Analysis Of Fatty Acids Research Articles

Quantitative trait locus analysis of unsaturated fatty acids in a recombinant inbred population of soybean

Soybean [Glycine max (L.) Merr.] is an important oilseed crop which produces about 30 % of the world’s edible vegetable oil. The quality of soybean oil is determined by its fatty acid composition. Soybean oil high in oleic and low in linolenic fatty acids is desirable for human consumption and other uses. The objectives of this study were to identify quantitative trait loci (QTLs) for unsaturated fatty acids and to evaluate the genetic effects of single QTL and QTL combinations in soybean. A population of recombinant inbred lines derived from the cross of SD02-4-59 × A02-381100 was evaluated for fatty acid content in seven environments. In total, 516 polymorphic single nucleotide polymorphism markers, 477 polymorphic simple sequence repeat markers and three GmFAD3 genes were used to genotype the mapping population. By using the composite interval mapping and/or the interval mapping method, a total of 15 QTLs for the three unsaturated fatty acids were detected in more than two environments. Two QTLs for oleic acid on linkage groups G [chromosome (Chr) 18] (qOLE-G) and J (Chr 16) (qOLE-J), three QTLs for linoleic acid on linkage groups A1 (Chr 5) (qLLE-A1) and G (Chr 18) (qLLE-G-1 and qLLE-G-2), and five QTLs for linolenic acid on linkage groups C2 (Chr 6), D1a (Chr 1), D1b (Chr 2), F (Chr 13) and G (Chr 18) were consistently detected in at least three individual environments and the average data over all environments. Significant QTL × QTL interactions were not detected. However, significant QTL × environment interactions were detected for all the QTLs which were repeatedly detected. Some QTLs reported previously were confirmed, and seven new QTLs (two for oleic acid, two for linoleic acid and three for linolenic acid) were identified in this study. Comparisons of two-locus and three-locus combinations indicated that cumulative effects of QTLs were significant for all the three unsaturated fatty acids. QTL pyramiding by molecular marker-assisted breeding would be an appropriate strategy for the improvement of unsaturated fatty acids in soybean.

Exploratory investigation reveals parallel alteration of plasma fatty acids and eicosanoids in coronary artery disease patients

Fatty acids and eicosanoids are two important classes of signaling lipid molecules involved in the pathogenesis of cardiovascular diseases. To investigate the physiological functions and interplay between fatty acids and eicosanoids in coronary artery disease (CAD) patients, we developed an analytical approach for parallel quantitative analysis of plasma fatty acids and eicosanoids, using gas chromatography–tandem mass spectrometry (GC–MS/MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS). In this study, 26 fatty acids and 12 eicosanoids were confidently detected in 12 patients with confirmed coronary artery disease and 11 healthy subjects. Pattern recognition analysis (principal components analysis, orthogonal partial least-square discriminate analysis, and hierarchical clustering analysis) demonstrated that the plasma lipid profile of fatty acids and eicosanoids enabled robust discrimination of CAD patients versus healthy subjects. Significant differences in six fatty acids and five eicosanoids were noted among CAD patients and healthy subjects. The development of cardiovascular disease-induced metabolic change of fatty acids and eicosanoids, such as eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid, hydroxyeicosatetraenoic acids and hydroxyoctadecadienoic acid, were consistent with previous isolated observations. Moderate-strong correlations between three plasma fatty acids and three eicosanoids from arachidonic acid metabolism were also observed. In brief, findings from this exploratory study offered a new insight on the roles of various bioactive lipid molecules in the development of coronary artery disease biomarkers.

Robotized method for the quantification of fatty acids by gas–liquid chromatography

Analysis of fatty acid profile in fats and oils is conventionally performed by gas–liquid chromatography (GLC) as their fatty acid methyl ester (FAME) derivatives. Accurate quantification can be achieved by adding internal standard(s) prior to the derivatization step and calibration of the system using certified FAME mixture. GLC instruments equipped with online sample preparation units became recently commercially available and in theory can be used to improve laboratory productivity and safety. However, studies showing that this type of approach can be used to obtain reliable results are scarce. In the present study, we developed a method suitable for quantitative analysis of fatty acids in fats and oils by robotic preparation and online GLC analysis of FAME using methanolic potassium hydroxide as transmethylation reagent. The method has been validated on different fats and oils including milk fat, palm kernel, rapeseed and long‐chain polyunsaturated fatty acids containing oil samples. Method performance has been compared to the corresponding manual procedure and results show good repeatability [CV(r)<5%] and good intermediate reproducibility [CV(iR)<5%] in accordance with FDA guidelines. The results obtained show that transmethylation and GLC analysis of fats and oils can be robotized while remaining accurate and providing fatty acid data expressed as g of fatty acid per 100 g of sample. Compared to conventional methods, the volume of reagent and solvent is three times lower per sample analyzed with the robotized version which contributes to the reduction of waste and chemical risk exposure.

QUANTITATIVE ANALYSIS OF FATTY ACIDS FROM SNOW LOTUS (SAUSSUREA) SPECIES USING HPLC WITH FLUORESCENCE DETECTION AND ATMOSPHERIC CHEMICAL IONIZATION-MASS SPECTROMETRY

Fatty acids (FA) are the basic and indispensable components in medicinal herbs. In the present work, a selective and sensitive method based on pre-column derivatization for the qualitative and quantitative analysis of FA from three snow lotus species (S. involucrata, S. laniceps, and S. medusa) was developed, and a comparative study based on the FA contents was carried out. FA were determined by HPLC with fluorescence detection, using 2-(12-oxobenzo[b]acridin-5(12H)-yl)-ethyl-4-toluenesulfonate (BAETS) as the labeling reagent and were identified with post-column APCI-MS. FA could be easily and quickly tagged by BAETS at 95°C in the presence of K2CO3 catalyst in N, N′-dimethylformamide (DMF). All validation procedures including linearity, limits of detection and quantification, precision, accuracy, ruggedness, and robustness were performed, and the results showed that the novel method exhibited excellent reproducibility and applicability. This proposed method was successfully applied to the analysis of total fatty acids (FA) and free fatty acids (FA) from snow lotus species. The content of FA in S. laniceps was significantly lower in comparison to the other two species that had similar FA content. Furthermore, this developed method exhibited a powerful potential for accurate detection of FA from other medicinal herbs or food stuff.

Quantitative trait locus analysis of saturated fatty acids in a population of recombinant inbred lines of soybean

Soybean [Glycine max (L.) Merr.] is an important crop which contributes approximately 58% of the world’s oilseed production. Palmitic and stearic acids are the two main saturated fatty acids in soybean oil. Different levels of saturated fatty acids are desired depending on the uses of the soybean oil. Vegetable oil low in saturated fatty acids is preferred for human consumption, while for industrial applications, soybean oil with higher levels of saturated fatty acids is more suitable. The objectives of this study were to identify quantitative trait loci (QTL) for saturated fatty acids, analyze the genetic effects of single QTL and QTL combinations, and discuss the potential of marker-assisted selection in soybean breeding for modified saturated fatty acid profiles. A population of recombinant inbred lines derived from the cross of SD02-4-59 × A02-381100 was grown in five environments and the seed samples from each environment were evaluated for fatty acid content. Genotyping of the population was performed with 516 polymorphic single nucleotide polymorphism markers and 298 polymorphic simple sequence repeat markers. Eight QTL for palmitic acid, five QTL for stearic acid and nine QTL for total saturated fatty acids were detected by composite interval mapping and/or interval mapping, with a high level of consistency or repeatability in multiple environments. Most of these QTL have not been reported previously, with the exception of qPAL-A1 which confirmed the result of a previous study. Significant QTL × QTL interactions were not detected. However, significant QTL × environment interactions were detected in most cases. Comparisons of two-locus and three-locus combinations indicated that cumulative effects of QTL were significant for both palmitic and stearic acids. QTL pyramiding by molecular marker-assisted selection would be an appropriate strategy for improvement of saturated fatty acids in soybean.

Rapid and direct quantitative analysis of positional fatty acids in triacylglycerols using 13C NMR

AbstractThere is increasing evidence that the positional fatty acid composition (FAC) of TAG is more important than total FAC with regard to nutrition values of edible oils and fats. A rapid and direct regiospecific analysis of positional fatty acids in TAG using 13C NMR was developed to overcome the tedious conventional methods which involve enzymatic hydrolysis, Grignard chemical degradation, and chromatography analysis. A set of NMR data acquisition parameters and processing methods had proven their excellent versatility and applicability on various types of oils and fats, with systematic error of 1.0 mol%. It was found that there are discrepancies between the regiospecific analysis results obtained by the current method and by conventional methods. Probable acyl migration occurring in the hydrolysis process in conventional methods is a noted problem. As the current 13C NMR method is a direct measurement and no hydrolysis of the sample is needed, acyl migration during the analysis is eliminated. As a result, the saturation level is always higher at sn‐1, 3 positions and lower at sn‐2 position in TAG structure of oils in the regiospecific data obtained from the current 13C NMR than that from conventional methods. In the absence of laborious chemical derivatization, this method is simple, accurate, and user‐friendly for researchers, especially for nutritionists to support their nutritional studies from the perspective of positional FAC of edible oils and fats.

Phytonutrient Profile of Purple Perilla (Perilla frutescens var. crispa) Seeds

To characterize phytonutrients, the seeds of 12 purple Perilla (Perilla frutescens var. crispa) accessions collected from Korea and Japan were used for quantitative analysis of tocopherols, phytosterols, squalene and fatty acids. The average tocopherol, squalene and phytosterols contents were 12.2 mg <TEX>$100g^{-1}$</TEX>, 3.99 mg <TEX>$100g^{-1}$</TEX> and 77.20 mg <TEX>$100g^{-1}$</TEX>, respectively. Among 4 tocopherol (T) isomers (<TEX>${\alpha}$</TEX>-T, <TEX>${\beta}$</TEX>-T, <TEX>${\gamma}$</TEX>-T, and <TEX>${\delta}$</TEX>-T), <TEX>${\gamma}$</TEX>-T was present in the highest quantity (11.03 mg <TEX>$100g^{-1}$</TEX>) with the least variation (CV = 13.7%), while <TEX>${\beta}$</TEX>-T was present in lowest quantity (0.25 mg 1<TEX>$100g^{-1}$</TEX>). Compared to campesterol (4.36 mg <TEX>$100g^{-1}$</TEX>) and stigmasterol (13.32 mg <TEX>$100g^{-1}$</TEX>), <TEX>${\beta}$</TEX>-sitosterol exhibited higher quantity (59.51 mg <TEX>$100g^{-1}$</TEX>) with 9.5% of variation. The major fatty acids were unsaturated fatty acids such as linolenic (61.5%), linoleic (17.3%), and oleic (9.9%) acids compared to saturated ones: palmitic (7.6%) and stearic (3.7%) acids. When Korean and Japanese accession were compared, almost no difference in content could be observed, while more variation as evaluated by CV (%) could be observed in Japanese accession in most phytonutrients suggesting wider genetic variation of purple Perilla in Japan. Presence of all above-mentioned phytonutrient compounds strongly suggested health beneficial value of purple Perilla seeds.

A new method for the determination of short-chain fatty acids from the aliphatic series in wines by headspace solid-phase microextraction–gas chromatography–ion trap mass spectrometry

A new analytical method for the determination of nine short-chain fatty acids (acetic, propionic, isobutyric, butyric, isovaleric, 2-methylbutyric, hexanoic, octanoic and decanoic acids) in wines using the automated HS/SPME–GC–ITMS technique was developed and optimised. Five different SPME fibers were tested and the influence of different factors such as temperature and time of extraction, temperature and time of desorption, pH, strength ionic, tannins, anthocyans, SO 2, sugar and ethanol content were studied and optimised using model solutions. Some analytes showed matrix effect so a study of recoveries was performed. The proposed HS/SPME–GC–ITMS method, that covers the concentration range of the different analytes in wines, showed wide linear ranges, values of repeatability and reproducibility lower than 4.0% of RSD and detection limits between 3 and 257 μg L −1, lower than the olfactory thresholds. The optimised method is a suitable technique for the quantitative analysis of short-chain fatty acids from the aliphatic series in real samples of white, rose and red wines.

Evaluation of two methods for plasma fatty acid analysis by GC

The present study was designed to establish the best method for the quantitative analysis of fatty acids in blood plasma. In the two steps method, internal standards of C15:0, C19:0, C19:1, or C21:0 fatty acids were added to plasma and lipids were extracted in chloroform/methanol (2:1). Lipid extracts resuspended in methanol/toluene (4:1) were subjected to methanolysis at 80°C for 2.5 h with acetyl chloride for GC analysis. In the direct method, plasma containing internal standards were suspended in methanol/toluene and after methanolysis at 100'C for 1 h with acetyl chloride were processed as above. When C 15:0 was used to calculate recoveries, values were above 100%, these being higher when using the direct method. When methylated C17:0 was the reference standard, the peak area ratios between internal standards and methylated reference were lower for C15:0 than for any of the others. When using C 19:0 as the internal standard, quantitative values of individual fatty acids were higher when the two steps method was used as compared to the direct method. Thus, the two steps method is more appropriate and reliable for the quantitative analysis of fatty acid profiles in plasma samples, and C19:0 but not C15:0 should be used as the internal standard.

Composition and authentication of virgin olive oil from French PDO regions by chemometric treatment of Raman spectra

AbstractThe search for the origin and the authentication of virgin olive oils (VOOs) is a problem of topical interest. VOOs with the protected designation of origin (PDO) label can be subject to fraudulent practices owing to high price commanded by these oils. This work evaluates the capability of confocal Raman spectroscopy combined with chemometric treatments for determining the fatty acid and triacylglycerol (TAG) compositions of VOOs and for authenticating the PDO labels of six French VOOs. Fatty acid and TAG compositions were determined by chemometric analysis of the Raman spectra using the partial least square (PLS) algorithms. Predictions have provided good results on the high percentage compounds, but for the fatty acids at very low percentages the predicted results were very poor. The PLS‐discriminant analysis model on Raman spectra was able to correctly classify 92.3% of French PDOs and 100% of PDO samples made with only one principal cultivar. However, it was noticeable that the first regression coefficient (which explains the greatest amount of variation of the data) obtained for each PDO could be compared one by one with the first regression coefficient obtained for the quantitative analysis of fatty acids and TAGs. The first regression coefficients obtained for PDOs differed widely from each other. Their study allowed interpreting the PDOs according to their chemical composition. This work confirms that fatty acid and TAG percentages may be used to authenticate French VOOs into PDOs. Copyright © 2011 John Wiley &amp; Sons, Ltd.

Typing of unknown microorganisms based on quantitative analysis of fatty acids by mass spectrometry and hierarchical clustering

Rapid identification of unknown microorganisms of clinical and agricultural importance is not only critical for accurate diagnosis of infections but also essential for appropriate and prompt treatment. We describe here a rapid method for microorganisms typing based on quantitative analysis of fatty acids by iFAT approach (Isotope-coded Fatty Acid Transmethylation). In this work, lyophilized cell lysates were directly mixed with 0.5 M NaOH solution in d3-methanol and n-hexane. After 1 min of ultrasonication, the top n-hexane layer was combined with a mixture of standard d0-methanol derived fatty acid methylesters with known concentration. Measurement of intensity ratios of d3/d0 labeled fragment ion and molecular ion pairs at the corresponding target fatty acids provides a quantitative basis for hierarchical clustering. In the resultant dendrogram, the Euclidean distance between unknown species and known species quantitatively reveals their differences or shared similarities in fatty acid related pathways. It is of particular interest to apply this method for typing fungal species because fungi has distinguished lipid biosynthetic pathways that have been targeted for lots of drugs or fungicides compared with bacteria and animals. The proposed method has no dependence on the availability of genome or proteome databases. Therefore, it is can be applicable for a broad range of unknown microorganisms or mutant species.

Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry

Sebum is a complex lipid mixture that is synthesized in sebaceous glands and excreted on the skin surface. The purpose of this study was the comprehensive detection of the intact lipids that compose sebum. These lipids exist as a broad range of chemical structures and concentrations. Sebum was collected with SebuTape(TM) from the foreheads of healthy donors, and then separated by HPLC on a C8 stationary phase with sub 2 µm particle size. This HPLC method provided high resolution and excellent reproducibility of retention times (RT). Compound mining was performed with time of flight (TOF) and triple quadrupole (QqQ) mass spectrometers (MS), which allowed for the classification of lipids according to their elemental composition, degree of unsaturation, and MS/MS fragmentation. The combination of the two MS systems detected 95 and 29 families of triacylglycerols (TAG) and diacylglycerols (DAG), respectively. Assignment was carried out regardless of positional isomerism. Among the wax esters (WE), 28 species were found to contain the 16:1 fatty acyl moiety. This method was suitable for the simultaneous detection of squalene and its oxygenated derivative. A total of 9 cholesterol esters (CE) were identified and more than 48 free fatty acids (FFA) were detected in normal sebum. The relative abundance of each individual lipid within its own chemical class was determined for 12 healthy donors. In summary, this method provided the first characterization of the features and distribution of intact components of the sebum lipidome.

Classification of diabetes deficiency syndromes based on plasma fatty acid metabolic profilings using pre-column derivatization quantitative method

A simple metabolic profiling approach for quantitative analysis of free fatty acids (FFAs) in human plasma by high performance liquid chromatography was described and validated, using alpha-bromoacetophenone as the derivative reagent and heptadecanoic acid (C(17:0)) as the internal standard. The quantitations of 6 predominant FFAs and 6 trace FFAs were achieved. Plasma fatty acid metabolic profiling of 75 diabetic patients was investigated, and then analyzed by multivariate statistical analysis. The linear discriminant analysis (LDA) model was established and validated for the pattern discrimination between Qi-deficiency and Qi and Yin-deficiency, with the hit ratio 94.3%. Stepwise discriminant analysis (SDA) model indicated that arachidonic acid (C(20:4)) and oleic acid (C(18:1)) contained the important information on the two syndromes above, and can be used as potential biomarkers of traditional Chinese medicine (TCM) syndrome. It is of great significance to systematically study the relationship between fatty acid metabolic profiling and TCM syndrome using metabolomics methods, and to improve the credibility and repeatability of clinical diagnosis and treatment system.

Omega-3 Fatty Acid Composition of Persian Gulf Fishes

The aim of this study was to investigate the content of omega-3 fatty acids in Persian Gulf fishes. The fishes were collected from Persian Gulf and the content of fatty acids in the head, muscle, and liver of fishes were determined. Quantitative analysis of fatty acids was performed by gas chromatography (GC) and methylmyristate was used as the reference material. GC and mass spectrometer (GC- mass) was applied for qualitative analysis and cod liver oil with all of omega-3 fatty acids was used as standard. Ghezel ala, Zamin kan-e-dom navari and Sorkhu mahi had maximum levels of omega-3 in total body. Halva Sefid, Gish-e-deraz baleh and Shamshiri were poor sources of omega-3. The liver of fish had the most content of omega-3 fatty acids followed by muscle and head, respectively.

Toxicity evaluation of Fibrocapsa japonica from the Northern Adriatic Sea through a chemical and toxicological approach

Since the 1990s red-tide blooms of Fibrocapsa japonica have frequently been observed in European waters; despite their recurrence also in the Adriatic coastal areas, they have never been conclusively linked to ichthyotoxic events. Since the toxicity of F. japonica is still under debate and its effects differ among the strains, in this study all the compounds previously postulated as involved in the toxic mechanism, such as brevetoxins, fatty acids and ROS, were screened for Adriatic strains. Whole algal extracts were analyzed for brevetoxin PbTx-2, which was not found, and for a qualitative and quantitative analysis of fatty acids. The fatty acid profile evidenced the presence of PUFAs, with considerable amounts of 18:4n-3, 20:4n-6, and 20:5n-3, as already found in different strains, but with a lower amount of arachidonic acid (20:4n-6). None of the PUFAs was released in the extracellular medium. Different toxicological assays ( Vibrio fischeri, Artemia sp., haemolysis of fish erythrocytes) were performed using algal cellular, extracellular or subcellular samples as well as fractions of the algal extract, in order to identify the toxic compounds. The tested cultures were found to inhibit V. fischeri bioluminescence, to affect Artemia nauplii viability and to have haemolytic effects comparable to those previously observed in different strains. The solid phase extraction (SPE) of the Adriatic F. japonica extracts resulted in a high concentration of PUFAs in two main fractions, which caused mortality in Artemia nauplii and inhibition of V. fischeri bioluminescence. Fish ( Dicentrarchus labrax) assays were also performed, reporting mortality after long exposure times and the cause of fish death was investigated. A significant increase of H 2O 2 in the tanks where sea basses were exposed to F. japonica was obtained, indicating that the presence of fish stimulates H 2O 2 production by algal cells and leading to the hypothesis of H 2O 2 involvement in F. japonica toxicity. The presence of oxidative stress in the exposed fish was confirmed by the increased malondialdehyde concentration in their gills. Overall these results indicate that since F. japonica cells can easily cling to fish gills, due to their high polysaccharide production, it is presumable that high PUFA amounts are released in loco, as a result of cell breakage, and that exert their toxicity together with ROS. This study shows that the Adriatic strains of F. japonica can be harmful to higher level organisms. Nevertheless a high cell density and a long lasting bloom are necessary to cause severe damage to fish gills or death for crustaceans.

Comparison between NIR, MIR, concatenated NIR and MIR analysis and hierarchical PLS model. Application to virgin olive oil analysis

This work investigates the potential use of simultaneously near-infrared (NIR) and mid-infrared (MIR) spectroscopies for the quantitative analysis of fatty acids and triacylglycerols and for identifying the Registered Designation of Origin (RDO) of extra virgin olive oils. The two spectral ranges were used separately using PLS and PLS-DA regressions. To combine both information, concatenated matrix was used at the first time, multiblock method using H-PLS models were constructed at the second time. The models were compared in terms of prediction errors. The results obtained with MIR spectroscopy are better than the ones obtained with NIR spectroscopy. The H-PLS methodology seems to be very interesting for quantitative analysis with the use of additional information in the NIR range, which is not present in the MIR one. For RDO identification by discriminant analysis, the use of multiblock method was less efficient.

低アレルゲン大豆「なごみまる」のイソフラボンおよび脂肪酸分析と「なごみまる」を原料とした味噌の官能評価

低アレルゲン大豆「なごみまる」のイソフラボンおよび脂肪酸分析と「なごみまる」を原料とした味噌の官能評価

Evaluation of a rapid method for the quantitative analysis of fatty acids in various matrices

A simplified method for quantitative analysis of fatty acids in various matrices by gas chromatography is proposed as an alternative to the conventional method and the variables of the protocol examined to optimize the processing conditions. The modified method involves direct methylation of fatty acids in homogenized samples with boron trihalide (BF 3 or BCl 3 in methanol) followed by extraction with hexane. The addition of hexane to the reaction mixture after the methylation process can enhance the efficiency of fatty acid methylation and is critical for those samples that contain high levels of triglycerides. A mechanism underlying this effect is proposed.

One-step method for quantitative and qualitative analysis of fatty acids in marine animal samples

In the one-step method, fatty acid methyl esters (FAMEs) have been prepared without prior lipid extraction. Each oyster, clam, and squid sample was esterified with 14% boron-trifluoride (BF 3) in methanol at 100 °C for 120 min, and hexane was used to help dissolve the lipids. This method gave a higher recovery of fatty acids as compared to the conventional method. Using an internal standard, the fatty acid content of the sample can be determined quantitatively and qualitatively. Due to its simplicity, speed, and reduced organic solvent usage, the one-step method should be useful for the determination of fatty acid composition in marine biological samples.

An extended chemical analysis of gallstone.

Chemical composition of gall stones is essential for aetiopathogensis of gallstone disease. We have reported quantitative chemical analysis of total cholesterol bilirubin, calcium, iron and inorganic phosphate in 120 gallstones from haryana. To extend this chemical analysis of gall stones by studying more cases and by analyzing more chemical constituents. A quantitative chemical analysis of total cholesterol, total bilirubin, fatty acids, triglycerides, phospholipids, bile acids, soluble proteins, sodium potassium, magnesium, copper, oxalate and chlorides of biliary calculi (52 cholesterol, 76 mixed and 72 pigment) retrieved from surgical operation of 200 patients from Haryana state was carried out. Total cholesterol as the major component and total bilirubin, phospholipids, triglycerides, bile acids, fatty acids (esterified), soluble protein, calcium, magnesium, iron, copper, sodium, potassium, inorganic phosphate, oxalate and chloride as minor components were found in all types of calculi. The cholesterol stones had higher content of total cholesterol, phospholipids, fatty acids (esterified), inorganic phosphate and copper compared to mixed and pigment stones. The mixed stones had higher content of iron and triglycerides than to cholesterol and pigment stones. The pigment stones were richer in total bilirubin, bile acids, calcium, oxalate, magnesium, sodium, potassium, chloride and soluble protein compared to cholesterol and mixed stones. Although total cholesterol was a major component of cholesterol, mixed and pigment gall stone in Haryana, the content of most of the other lipids, cations and anions was different in different gall stones indicating their different mechanism of formation.