BiotecVisions 2012, April

The natural source of human milk fat substitute (HMFS) is a field worth exploring. In this study, tilapia oil was extracted and analyzed. In the triacylglycerol fraction, the contents of sn-2 palmitic acid and total sn-1,3 oleic acid and linoleic acid were 48.01% and 66.62%, respectively. The optimal solvent fractionation conditions were determined to be a tilapia oil-to-acetone ratio of 1:8 (w/v), crystallization temperature of -30°C, and crystallization duration of 16h, giving a solid fraction yield of 64.20%. In fractionated tilapia oil, the total content of 1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) and 1,3-dioleoyl-2-palmitoylglycerol (OPO) increased by 20.38%, as determined by reversed-phase liquid chromatography. Ultra-high-performance combined-phase chromatography combined with quadrupole time-of-flight mass spectrometry analysis showed that OPL (17.45%) was the most abundant triacylglycerol in fractionated tilapia oil, followed by OPO (13.90%). Fractionated tilapia oil is thus an excellent source of OPL and has great potential for incorporation in HMFS. PRACTICAL APPLICATION: Human milk fat substitutes are an important component of infant formulas. This work provides an excellent natural source of oil rich in OPL, which has great potential in the field of preparing human milk fat substitutes highly similar to human milk fat.

Fatty acid composition and distribution of human milk fat (HMF), from mothers over different lactating periods in Guangzhou, China, were analyzed. The universal characteristics were consistent with previously reported results although the fatty acid content was within a different range and dependent on the local population (low saturated fatty acid and high oleic acid for Guangdong mothers' milk fat). Based on the composition of the total and sn-2 fatty acids of mature milk fat, an efficient evaluation model was innovatively established by adopting the "deducting score" principle. The model showed good agreement between the scores and the degree of similarity by assessing 15 samples from different sources including four samples of HMF, eight samples of human milk fat substitutes (HMFSs) and infant formulas, and three samples of fats and oils. This study would allow for the devolvement of individual human milk fat substitutes with different and specific fatty acid compositions for local infants.

Being the dominant components in human milk fat (HMF), triacylglycerol (TAG) composition might be the best approximation index to represent the composing characteristics of HMF. In this study, TAG composition of HMF from different lactation stages was analyzed by RP-HPLC-APCI-MS, and the establishment of a model for the precise evaluation of human milk fat substitutes (HMFSs) based on TAG composition was indirectly realized by employment of fatty acid composition and distribution and polyunsaturated fatty acid (PUFA) and TAG compositions. The model was verified by the selected fats and oils with specific chemical compositions, and the results revealed the degrees of similarity of these fats and oils in different evaluation aspects reflected their differences in corresponding chemical composition with HMF. The newly established evaluation model with TAG composition as a comparison base could provide a more accurate method to evaluate HMFSs and might have some inspirations for HMFS production in the future.

The production of triacylglycerols (TAGs) with similar structures to the human milk fat TAGs is only possible using sn‐1,3 regioselective lipases as biocatalysts. The advances on the processes currently performed for the production of human milk fat substitutes (HMFS), namely the biocatalysts used, the best operation conditions, reactor types and operation modes, as well as the major drawbacks of these enzymatic systems, are addressed in this article.

Human milk fat substitute (HMFS), one of the vital ingredients of infant formula, constitutes about 50% of energy intake for many infants. The composition of HMFS depends on the raw material and the production technology. Both human and animal studies have shown that absorption of fats and minerals are affected by triacylglycerol structures and fatty acid compositions of HMFS. Additionally, the level of other micronutrients, such as fat‐soluble vitamins, can also be affected by production processes. The ultimate goal in the design of infant formula is to mimic human milk, regarding both lipids and other micronutrients. This paper covers human milk fat and selected HMFS as well as their impact on absorption of fat and calcium and illustrates recent progresses and findings.

CXCR4 is a chemokine receptor specific for stromal derived factor-1 (SDF-1) and is highly expressed in breast cancer cell lines and primary breast cancer samples. Elevated expression of CXCR4 in breast cancer is linked to poor prognosis. CXCR4 signalling has been reported to be important in breast cancer migration and metastasis formation but its role is yet to be fully elucidated. Our previously published data have shown that a stem cell-enriched population of cells can be obtained by harvesting anoikis resistant cells which survive in early mammosphere culture (Harrison et al., Cancer Res; 2010, 70 (2), 709-18). We collected anoikis resistant cells 12 hours after plating out into the mammosphere culture to obtain stem cell-enriched populations from 2 immortalised (MCF10A, 226L) and 3 malignant breast cell lines (MCF7, T47D, SKBR3). Anoikis resistant cells harvested at 12 hours had a significantly higher mammosphere colony-forming efficiency than their unsorted monolayer cells (2-4 fold increase, p < 0.05) suggesting stem cell enrichment. In limiting dilution transplantation assays into athymic Nude mice, MCF7 and T47D anoikis resistant cells had a 12 and 16-fold enrichment for tumor formation respectively, compared to the unsorted monolayer cells. Using an Agilent custom microarray, we found that CXCR4 expression was consistently upregulated in these stem cell-enriched populations compared with monolayer cells (2.0 and 6.8 fold; normal and malignant cell line averages respectively). We further investigated the function of CXCR4 in vitro using the mammosphere assay to assess stem cell activity. AMD3100 (Sigma), a CXCR4 antagonist and lentiviral CXCR4 shRNA were used to inhibit and recombinant soluble SDF-1 to stimulate the pathway. In the normal breast cell line MCF10a, SDF-1 addition significantly reduced mammosphere formation in a dose dependent manner (200ng/ml; 25% decrease, t = 4.320, p < 0.001) and this could be rescued by the addition of AMD3100. Inhibiting the CXCR4 pathway stimulated primary mammosphere formation in the breast cancer cell lines T47D and MCF7 using either 20uM AMD3100 (T47D; 34% increase, t = 3.017, p < 0.01) or lentiviral CXCR4 shRNA (T47D; 97% increase, t = 6.288 p < 0.005, MCF7; 48% increase, t = 3.026, p < 0.05). In vivo data has already shown the importance of the CXCR4/SDF-1 pathway in metastasis formation in breast cancer. Our data takes suggests that CXCR4 signalling, as well as its role in migration and homing, is important in maintaining stem cell activity. Thus CXCR4 targeted therapy, alongside current standards of care, may improve breast cancer outcomes. Funding: Biotechnology and Biological Sciences Research Council (BBSRC), Vertex Pharmaceuticals (Europe) Limited, Breast Cancer Campaign and Cancer Research UK (CRUK). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3339. doi:10.1158/1538-7445.AM2011-3339

The effect of fatty acid positioning in dietary triacylglycerols and intake of long-chain n-3 polyunsaturated fatty acids on bone mineral accretion in growing piglets

In this study, the influence of total sn-2 palmitic triacylglycerols (TAGs) and ratio of 1-oleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) to 1,3-dioleoyl-2-palmitoylglycerol (OPO) in human milk fat substitute (HMFS) on the metabolic changes were investigated in Sprague-Dawley rats. Metabolomics and lipidomics profiling analysis indicated that increasing the total sn-2 palmitic TAGs and OPL to OPO ratio in HMFS could significantly influence glycine, serine and threonine metabolism, glycerophospholipid metabolism, glycerolipid metabolism, sphingolipid metabolism, bile acid biosynthesis, and taurine and hypotaurine metabolism pathways in rats after 4 weeks of feeding, which were mainly related to lipid, bile acid and energy metabolism. Meanwhile, the up-regulation of taurine, L-tryptophan, and L-cysteine, and down-regulations of lysoPC (18:0) and hypoxanthine would contribute to the reduction in inflammatory response and oxidative stress, and improvement of immunity function in rats. In addition, analysis of targeted biochemical factors also revealed that HMFS-fed rats had significantly increased levels of anti-inflammatory factor (IL-4), immunoglobulin A (IgA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px), and decreased levels of pro-inflammatory factors (IL-6 and TNF-α) and malondialdehyde (MDA), compared with those of the control fat-fed rats. Collectively, these observations present new in vivo nutritional evidence for the metabolic regulatory effects of the TAG structure and composition of human milk fat substitutes on the host.

A standard treatment for cancer often involves the use of adjuvant supplementation with targeted therapy to maximize tumor suppression. Recent studies have shown that docosahexaenoic acid (DHA; C22:6 n-3), a long chain omega-3 polyunsaturated fatty acid, acts on an array of molecular oncogenes that are often targeted by anti-tumorigenic drugs. After a careful review of DHA’s mechanistic properties, 2-deoxyglucose (2DG), metformin, and aspirin were identified as drugs that share similar molecular targets as DHA. 2DG and metformin both target cellular metabolism by inhibiting intracellular ATP, albeit different mechanisms. 2DG inhibits glycolysis, while metformin targets the mitochondria. Aspirin inhibits inflammation via irreversible acetylation of platelet COX. The current study investigates the effects of DHA in combination with each drug on numerous cancer subtypes to determine if concurrent treatments can synergistically inhibit tumor proliferation. In BT-474 human breast ductal carcinoma, MDA-MB-231 human breast adenocarcinoma, and A549 human lung adenocarcinoma, DHA was shown to enhance the tumor-inhibiting effects of 2DG via intracellular ATP reduction. In contrast, DHA showed no effect with metformin on the same tumor cell lines. With limited studies localizing DHA’s metabolic effects, these results suggest that DHA may have potential selectivity for ATP inhibition via glycolysis. In response to DHA + aspirin, the PC-3 prostate adenocarcinoma and MDA-MB-231 displayed synergistic inhibition of cell viability, while LNCaP prostate carcinoma showed an additive effect. With studies showing DHA’s ability to inhibit endothelial COX, further studies will be done to determine whether the synergistic effects of DHA + aspirin expand to COX expression as well. Moreover, downstream targets of 2DG and metformin will be studied to determine the extent of DHA enhancement on oncogenic targets, such as mTOR, 4EB-P1, and GRB10. The results of this study provide preliminary evidence for the efficacy of adjuvant supplementation with DHA in combination with anti-tumorigenic drugs. With ongoing analysis, the potential for adjuvant nutritional supplementation will allow for additional treatment strategies for future patients. Citation Format: Irvin V. Ma, Michael Mouradian, Andrew R. Cooper, Erika D. Vicente, Ronald S. Pardini. Adjuvant supplementation of docosahexaenoic acid with 2-deoxyglucose, metformin, and aspirin on cellular metabolism in breast, lung, and prostate tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1616.

Lipase-mediated human milk fat substitute production: Mechanistic insights and a rational synthesis strategy.

Human milk fat substitute (HMFS) is a fat that contains 1,3-dioleoyl-2-palmitoyl-glycerol (OPO) as the main triacylglycerol. Hard palm stearin (HPS) is a palm oil fraction containing high palmitic acid (PA), which can be used as a raw material in OPO synthesis. The challenge in OPO synthesis using HPS is to maintain sn-2 PA and increase sn-1,3 oleic acid (OA). This research was conducted to synthesize OPO through enzymatic acidolysis of the HPS-solid fraction and OA (1:6, mole ratio) using Lipozyme TL IM 10% of the substrate weight at 60 °C for 2 – 24 h. The results showed that acidolysis caused increased partial acylglycerol, decreased TAG, and decreased sn-2 PA while increased sn-2 OA and sn-1,3 OA. The presence of acyl migration caused the incorporation of OA using Lipozyme TL IM at the sn-1,3 positions after 2 h was not significantly different. The product obtained at 2 h contained TAG with 74% sn-2 PA and 17% sn-2 OA, 78% sn-1,3 PA, and 11% sn-1,3 OA.

The aim of this work was to investigate the catalytic functions of a new immobilized Thermomyces lanuginosa lipase in interesterification and to optimize the conditions of interesterification for the production of human milk fat substitutes (HMFS) containing n−3 PUFA by response surface methodology (RSM). Thermomyces lanuginosa lipase had an activity similar to that of immobilized Rhizomucor miehei lipase (Lipozyme RM IM) in the glycerolysis of sunflower oil, but the former had higher activity at a low reaction temperature (5°C). Thermomyces lanuginosa lipase was found to have much lower catalytic activity than Lipozyme RM IM in the acidolysis of sunflower oil with caprylic acid. However, the activity of T. lanuginosa lipase was only slightly lower than that of Lipozyme RM IM in the ester‐ester exchange between tripalmitin (PPP) and the ethyl esters of EPA and DHA (EE). For this reason, the new immobilized T. lanuginosa lipase was used to produce HMFS from PPP by interesterification with EE. The optimization of major parameters was conducted with the assistante molar ratio of 5 (EE/PPP), a lipase load of 20 wt% (on substrates), and a reaction time of 20 h, with acyl incorporation up to 42%. The model generated significantly represented real relationships between the response (incorporation) and reaction parameters.

The aim of this paper was the analytical evaluation of human milk fat substitutes (HMFS) by the calorimetric and spectroscopic methods. The HMFS were obtained by enzymatic interesterification of blend of lard or milk fat with rapeseed oil and concentrate of fish oil. The enzymatic reactions were carried out at 60, 70, and 80 °C for 2 h. A commercially immobilized 1,3-specific lipase, Lipozyme RM IM, was used as a biocatalyst. Oxidative stability of HMFS was determined using the calorimetric method. The oxidative induction time was obtained from the pressure differential scanning calorimetry curves. Peroxide value (PV) and anisidine value were determined using spectroscopic method. Interesterification caused a decrease in oxidative stability. Samples with lower induction times were characterized by higher PV. There was also a strong relation between total polar compound content and induction time. The induction times obtained for analyzed fats can be used as primary parameters for the assessment of the resistance of tested fats to their oxidative decomposition.

A novel and controllable method for simultaneous preparation of human milk fat substitutes (OPL, OPO and LPL): two-step enzymatic ethanolysis-esterification strategy

12005 Background: Numerous pharmacogenetic studies have led to the identification of genetic polymorphisms associated not only with the development of cardiovascular disease, but also increase the risk of complications due to the use of anthracycline drugs, widely used in the treatment of cancer. The purpose of this study was to study the frequency of rs4673 and rs28714259 and possible associations with the risk of cardiovascular changes in patients with breast cancer during anthracycline therapy (anthracycline-mediated cardiotoxicity — AMC). Methods: The study included 256 Caucasian patients (median age - 55 years) with a diagnosis of breast cancer without diagnosed cardiovascular changes, who were treated with anthracyclines at the National Medical Research Center of Oncology in 2019-2020. For genotyping of rs4673 and rs28714259, DNA was extracted from blood using DNA-sorb-B (AmpliSens, Russia) and HRM-PCR was performed on a CFX96 amplifier (Bio-Rad, USA). The presence of polymorphisms was confirmed by Sanger sequencing on a Genetic Analyzer 3500 (ABI, USA). Results: During the follow-up period 21 (8.2%) patients were diagnosed with signs of subacute (changes developed within several weeks after the last course of therapy) or early chronic AMC (changes developed within a year after completion of anthracycline therapy). In the group of patients without AOC the allelic frequency of rs4673 (c.214T > C CYBA) was 0.38, the frequency of genotypes C/C – 0.4, C/T – 0.43, and T/T – 0.17. In the same group, the frequency of the A allele rs28714259 was 0.07, the frequency of the G/G genotypes – 0.87, G/A – 0.13, and A/A – 0. The prevalence of genotypes T/T rs4673 and allele G rs28714259 in a cohort of Russian patients differed from the European population (p = 0.014 and p = 0.05, respectively). The risk of cardiovascular changes on the background of anthracycline therapy increased in the presence of the rs4673 polymorphic allele by 6.49 times, in the case of the G/A and A/A rs28714259 genotypes - by 3.27 times. The results of the ROC-analysis suggested high quality of the tests based on the dominant models rs4673 and rs28714259 (AUC was 71.9% and 76.3% correspondingly). Conclusions: In this study the prognostic efficiency of the genetic markers rs4673 and rs28714259 was shown for the prompt detection of the risks of AMC development in the management of cancer patients. However, population characteristics should be taken into consideration for risk assessment.