Een investigated. The objective of this study was to determine the effects of HIV-1 infection and viral subtype on patient’s cholesterol (TC, LDLC, HDLC), lipid peroxidation indices (LPI), and oxidative stress markers [total antioxidant ability (TAA) and malondialdehyde (MDA)], to determine whether in the absence of HAART, these biochemical parameters could be useful in patient’s management and monitoring disease progression.Sample CollectionFollowing a 10 hour fast, 10 ml of blood was collected from each participant into a Rubusoside labeled dry tube (5 ml) and a tube containing EDTA anticoagulant (5 ml). Following clotting, the dry tubes were centrifuged at 1200 g for 15 min to collect serum which was aliquoted and used for the assay of the different biochemical parameters while EDTA tubes were centrifuged at the same conditions to collect plasma which was also aliquoted and used for RNA extraction and measurement of oxidative stress markers. All samples were stored at 220uC and processed within four days after collection. 1) Quantification of biochemical parameters. Serum TC, HDLC, LDLC concentrations were determined using commercially available kits (Human Gesellschaft fur Biochemica und DiagnosticambH Kit, Max-Planck-Ring 21-Wiesbaden-Germany). Plasma TAA was determined using the method of Benzie and Naringin biological activity Strain [21], while MDA was 16985061 determined using the method of Kohn and Liversedge [22] as described by Lefevre [23]. MDA ` measurement is based on a reaction between malondialdehyde and thiobarbituric acid which forms a pink pigment that absorbs at 532 nm at 90uC 2100uC at pH 2, while TAA is measured using the ferric reducing ability of plasma (FRAP) based on the ability of the antioxidants present in plasma to reduce Fe3+ to Fe2+ at pH 3.6 in the presence of 2.4.6 tri (22 pyridyl-s-triazine); the reaction produces an intense blue color that absorbs at 593 nm. LPI was obtained by calculating the ratio MDA/TAA. TC concentration was determined using colorimetric enzymatic techniques based on the successive action of cholesterol oxidase and peroxidase; HDLC concentration in the serum supernatant was determined by the same process after the precipitation of VLDL cholesterol, LDL 23148522 cholesterol and chylomicrons in the presence of phosphotungstic acid and MgCl2. Results were calculated using the formula: TC (g/l) or HDLC (mg/dl) concentrations = (OD500nm sample/ OD500nm standard) 6 Concentration of standard (essentially as recommended by the manufacturer in the kits). LDLC concentration was determined using the formula of Friedewaldet al. [24]: LDLC (mg/dl) = TC (mg/dl)-[HDLC (mg/dl)-Triglycerides (mg/dl)/5]. 2) RNA extraction and PCR amplification. RNA was extracted from patient’s plasma using the QIAmp Viral RNA kit ([Qiagen, Hilden, Germany]), according to the manufacturer’s instructions. RNA and cDNA samples were then amplified in a one tube, two steps RT- PCR using a thermal cycler (TECHNE TC 412[TECHNE Inc, Burlington, New Jersey, USA]) with the following primers: H1G777 (TCACCTAGAACTTTGAATGCATGGG) sense (nucleotide 777 to 801 of HIV-1 genome) and H1P202 (CTAATACTGTATCATCTGCTGCTCCTGT) antisense (nucleotide 1874 to 1898 of HIV-1 genome). Nested PCR was performed using H1Gag1584 (AAAGATGGATAATCCTGGG) sense and g17 (TCCACATTTCCAACAGCCCTT) antisense primers to enable amplification of the 460 bp encoding amino acid 132 of p24 to amino acid 40 of p7 from the gag gene. The amplification conditions of the RT-PCR were as followed: 50 min at 42uC (cDNA reaction).Een investigated. The objective of this study was to determine the effects of HIV-1 infection and viral subtype on patient’s cholesterol (TC, LDLC, HDLC), lipid peroxidation indices (LPI), and oxidative stress markers [total antioxidant ability (TAA) and malondialdehyde (MDA)], to determine whether in the absence of HAART, these biochemical parameters could be useful in patient’s management and monitoring disease progression.Sample CollectionFollowing a 10 hour fast, 10 ml of blood was collected from each participant into a labeled dry tube (5 ml) and a tube containing EDTA anticoagulant (5 ml). Following clotting, the dry tubes were centrifuged at 1200 g for 15 min to collect serum which was aliquoted and used for the assay of the different biochemical parameters while EDTA tubes were centrifuged at the same conditions to collect plasma which was also aliquoted and used for RNA extraction and measurement of oxidative stress markers. All samples were stored at 220uC and processed within four days after collection. 1) Quantification of biochemical parameters. Serum TC, HDLC, LDLC concentrations were determined using commercially available kits (Human Gesellschaft fur Biochemica und DiagnosticambH Kit, Max-Planck-Ring 21-Wiesbaden-Germany). Plasma TAA was determined using the method of Benzie and Strain [21], while MDA was 16985061 determined using the method of Kohn and Liversedge [22] as described by Lefevre [23]. MDA ` measurement is based on a reaction between malondialdehyde and thiobarbituric acid which forms a pink pigment that absorbs at 532 nm at 90uC 2100uC at pH 2, while TAA is measured using the ferric reducing ability of plasma (FRAP) based on the ability of the antioxidants present in plasma to reduce Fe3+ to Fe2+ at pH 3.6 in the presence of 2.4.6 tri (22 pyridyl-s-triazine); the reaction produces an intense blue color that absorbs at 593 nm. LPI was obtained by calculating the ratio MDA/TAA. TC concentration was determined using colorimetric enzymatic techniques based on the successive action of cholesterol oxidase and peroxidase; HDLC concentration in the serum supernatant was determined by the same process after the precipitation of VLDL cholesterol, LDL 23148522 cholesterol and chylomicrons in the presence of phosphotungstic acid and MgCl2. Results were calculated using the formula: TC (g/l) or HDLC (mg/dl) concentrations = (OD500nm sample/ OD500nm standard) 6 Concentration of standard (essentially as recommended by the manufacturer in the kits). LDLC concentration was determined using the formula of Friedewaldet al. [24]: LDLC (mg/dl) = TC (mg/dl)-[HDLC (mg/dl)-Triglycerides (mg/dl)/5]. 2) RNA extraction and PCR amplification. RNA was extracted from patient’s plasma using the QIAmp Viral RNA kit ([Qiagen, Hilden, Germany]), according to the manufacturer’s instructions. RNA and cDNA samples were then amplified in a one tube, two steps RT- PCR using a thermal cycler (TECHNE TC 412[TECHNE Inc, Burlington, New Jersey, USA]) with the following primers: H1G777 (TCACCTAGAACTTTGAATGCATGGG) sense (nucleotide 777 to 801 of HIV-1 genome) and H1P202 (CTAATACTGTATCATCTGCTGCTCCTGT) antisense (nucleotide 1874 to 1898 of HIV-1 genome). Nested PCR was performed using H1Gag1584 (AAAGATGGATAATCCTGGG) sense and g17 (TCCACATTTCCAACAGCCCTT) antisense primers to enable amplification of the 460 bp encoding amino acid 132 of p24 to amino acid 40 of p7 from the gag gene. The amplification conditions of the RT-PCR were as followed: 50 min at 42uC (cDNA reaction).
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