Patients with SCD (n = 47) and healthy controls (n = 46) aged 20 to 59 years were enrolled in the present study. Participants with HbSS or HbSβ0 genotypes were included as patients with SCD, and hemoglobin patterns were confirmed by high-performance liquid chromatography (HPLC) (Bio-Rad) and DNA sequence analysis. Patients who had received blood transfusions in the past 3 months, in vaso-occlusive crises and with apparent infection were excluded from the study. No patient was being treated with antibiotics or corticosteroids, and all patients were under treatment with folic acid, calcium, and vitamin D. Moreover, most patients were under treatment with hydroxyurea. Blood samples were obtained from the participants during regular consultation at the Unicamp Hematology and Hemotherapy Center, São Paulo, Brazil. Complete blood counts with reticulocyte counts were performed on blood collected with EDTA in a hematology analyzer (Beckman Coulter). The study was approved by the Unicamp Human Research Ethics Committee (protocol number CAAE: 85061318.0.0000.5404). All patients and controls had agreed to participate and had signed informed consent forms.
High performance liquid chromatography (hplc)
Manufactured by Bio-Rad
Sourced in United States, Germany, Japan
HPLC (High-Performance Liquid Chromatography) is a powerful analytical technique used for the separation, identification, and quantification of various chemical compounds in complex mixtures. It operates by pumping a liquid mobile phase through a stationary phase packed in a column, allowing the components of the mixture to be separated based on their interactions with the stationary phase. HPLC systems typically consist of a solvent delivery system, an injector, a separation column, a detector, and a data acquisition system.
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Lab products found in correlation
C8000, by Abbott (2 mentions)
Advia 1200, by Siemens (2 mentions)
Cobas 801e, by Roche (2 mentions)
Hpx 87h column, by Bio-Rad (2 mentions)
Accu check meter, by Roche (2 mentions)
Automated hematology analyzer, by Sysmex (2 mentions)
Cobas 8000, by Roche (2 mentions)
Hematology analyzer, by Beckman Coulter (1 mentions)
Porapak n column, by Shimadzu (1 mentions)
Gc tcd, by Shimadzu (1 mentions)
Rompun, by Bayer (1 mentions)
Zoletil, by Virbac (1 mentions)
C57blks j db m and db db mice, by Jackson ImmunoResearch (1 mentions)
2414 refractive index detector, by Waters Corporation (1 mentions)
Modular analyzer, by Roche (1 mentions)
Dako insulin elisa, by Agilent Technologies (1 mentions)
Vertex 70, by Bruker (1 mentions)
Ph meter, by HACH (1 mentions)
Au2700, by Olympus (1 mentions)
Eclia, by Roche (1 mentions)
66 protocols using high performance liquid chromatography (hplc)
1
Sickle Cell Disease Hematological Profile
2
Anaerobic Wastewater Treatment Evaluation
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Mixed liquor total and volatile suspend solids (MLSS, MLVSS, COV = ± 5%) and COD (COV = ± 5%) were measured according to the standard methods for the examination of water and wastewater (APHA, 2005 ). VFAs were measured on Shimadzu HPLC using a Biorad‐Aminex column, and the carrier solvent was 0.01 M H2SO4 at a flow rate of 0.7 ml min−1 at 60°C (COV = ± 8%). The composition of biogas was determined using a Shimadzu GC‐TCD fitted with a Porapak N column (1500 × 6.35 mm; Vyrides, 2009 ).
3
Anthocyanin Intervention in Diabetic Mice
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Male 6-week-old C57BLKS/J db/m and db/db mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). The mice were fed a regular chow diet, provided with water ad libitum, and allowed to acclimatise for 1 week before experiments. The mice were divided into four groups. Control db/m mice (n = 6) and control db/db mice (n = 6) received drinking water only, while anthocyanin db/m mice (n = 8) and anthocyanin db/db mice (n = 8) received 10 mg/kg body weight anthocyanin-rich SE daily for 12 weeks. For measurement of 24-h urinary albumin, the mice were placed in individual mouse metabolic cages (Nalgene, Rochester, NY, USA) every 4 weeks. At week 20, the mice were anaesthetised by intraperitoneal injection of a mixture of Rompun (10 mg/kg; Bayer Korea, Ansan, Gyeonggi-Do, Korea) and Zoletil (30 mg/kg; Virbac, Carros, France). Blood was collected from the left ventricle and centrifuged, and the resulting plasma was stored at −70°C for analyses. The kidneys were rapidly dissected and stored in 10% buffered formalin for immunohistochemical analyses. HbA1c was measured from red cell lysates by HPLC (Bio-Rad, Richmond, CA, USA). Triglyceride and total cholesterol concentrations were determined using an automatic analyser (Model 917; Hitachi, Tokyo, Japan) and commercial kits (Wako, Osaka, Japan). NEFA levels were measured with a JCA-BM1250 automatic analyser (JEOL, Tokyo, Japan).
4
HPLC Analysis of Glucose and Formate
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Glucose and formate were assayed by HPLC (Sykam, Gilching, Germany), equipped with
an HPX-87 P ion-exchange column Aminex (Bio-Rad Lab, USA). The mobile phase was 5
mM H2SO4 with the flow rate 0.8 mL/min. The column was
maintained at room temperature and the absorbance was measured at 210 nm. All
samples were filter-sterilized (0.2 μm pore size) to remove cells and other
particles before analysis.
an HPX-87 P ion-exchange column Aminex (Bio-Rad Lab, USA). The mobile phase was 5
mM H2SO4 with the flow rate 0.8 mL/min. The column was
maintained at room temperature and the absorbance was measured at 210 nm. All
samples were filter-sterilized (0.2 μm pore size) to remove cells and other
particles before analysis.
5
Biomarker Measurement in Diabetes
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Fasting plasma glucose (FPG) was quantified by the glucose oxidase procedure and HbA1c was measured by ion-exchange high-performance liquid chromatography (HPLC, Bio-Rad, USA). Creatinine (Cr) and uric acid (UA) were measured by an enzymatic method with a chemical analyzer (Hitachi 7600-020, Tokyo, Japan); CCR was calculated using the Cockcroft-Gault formula. The chemiluminescence-based immunoanalytical system was used to determine plasma levels of NT-proBNP (VITROS 5600 integrated system, Johnson & Johnson Medical Company, USA).
6
Comprehensive Metabolic and Biochemical Profiling
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A 12-h overnight fasting venous blood sample was collected in all subjects. A first morning urine sample was collected once a day for 3 consecutive days to estimate the Alb/Cr. The calcium, phosphate, uric acid, creatinine, albumin, total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and fasting plasma glucose (FPG) were measured by biochemical auto analyzer (Abbott C8000). Measurements of insulin and NT-proBNP were performed by immunoassay technique on the Roche Elecsys 2010 systerm. HbA1c was measured by high performance liquid chromatography (HPLC; Bio-Rad, Hercules, CA, USA). Serum calcium level was corrected according to the formula: albumin-adjusted serum calcium concentration (mg/dL) = measured serum calcium concentration (mg/dL) + 0.8 × [4 − serum albumin concentration (g/dL)] [10 (link)]. Insulin resistance was assessed by the homeostatic model: HOMA-IR = fasting plasma glucose (mmol/L) × fasting plasma insulin (mIU/L)/22.5 [16 (link)].
7
Quantification of Glucose and Isopentenol
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For glucose analysis, each culture sample was pelleted by centrifugation and the collected supernatant was analyzed in Waters HPLC equipped with a Bio-Rad Aminex HPX-87H Column (300 × 7.8 mm). The eluent (5 mM H2SO4) was pumped at a flow rate of 0.4 mL min−1. The column temperature was maintained at 55°C and the peaks were detected using a Waters 2414 refractive index detector.
For isopentenol analysis, each sample was prepared by ethyl acetate extraction and only the upper layer was subject to GC analysis. The GC equipped with a flame ionization detector (FID) and a CP-FFAP CB capillary column (50 m × 0.25 mm; 0.2 μm film thickness) was applied. The oven temperature was initially held at 50°C for 1 min, then raised with a gradient of 5°C/min until reaching 100°C, and finally programmed to 150°C at 25°C/min, then hold for 5 min. Nitrogen was used as the carrier gas. The injector and detector were held at 250°C and 270°C, respectively. This method can separate isoprenol and prenol efficiently, the isoprenol peak appeared at 13.9 min while the prenol peak appeared at 15.3 min.
For isopentenol analysis, each sample was prepared by ethyl acetate extraction and only the upper layer was subject to GC analysis. The GC equipped with a flame ionization detector (FID) and a CP-FFAP CB capillary column (50 m × 0.25 mm; 0.2 μm film thickness) was applied. The oven temperature was initially held at 50°C for 1 min, then raised with a gradient of 5°C/min until reaching 100°C, and finally programmed to 150°C at 25°C/min, then hold for 5 min. Nitrogen was used as the carrier gas. The injector and detector were held at 250°C and 270°C, respectively. This method can separate isoprenol and prenol efficiently, the isoprenol peak appeared at 13.9 min while the prenol peak appeared at 15.3 min.
8
Cardiometabolic Biomarkers in Fasting Samples
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A 12-hour overnight fasting venous blood sample was collected in all subjects. A morning fasting spot urine sample was collected once every month for three consecutive months to estimate the urinary albumin-to-urinary creatinine ratio. The creatinine, albumin, calcium, phosphate, uric acid, total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and fasting plasma glucose (FPG) were measured by biochemical auto analyzer (Abbott C8000). The fasting insulin concentration was measured by electrochemiluminescence immunoassay technique (Roche Elecsys 2010). HbA1c was measured by high performance liquid chromatography (HPLC; Bio-Rad, Hercules, CA, USA). Serum calcium level was corrected according to the formula: albumin-adjusted serum calcium concentration (mg/dl) = measured serum calcium concentration (mg/dl) + 0.8 × [4 − serum albumin concentration (g/dl)] [11 (link)]. Insulin resistance was determined using the homeostatic model assessment: HOMA-IR = fasting plasma glucose (mmol/l) × fasting plasma insulin (mIU/L)/22.5 [13 (link)].
9
Comprehensive Metabolic Marker Measurements
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Plasma glucose concentration was measured with a Yellow Springs glucose analyzer (YSI Inc). HbA1c was measured by HPLC (Bio-Rad). Plasma insulin was measured with Dako Insulin ELISA (DAKO) using a spectrophotometric analyzer. Glucagon concentration was measured with a Millipore Glucagon RIA Kit (Millipore Corporation).
The 2H atom percentage excess in plasma glucose was determined using a Thermo “Voyager” single quadruple mass spectrometer with Thermo “Trace” gas chromatograph (Thermo Scientific). Plasma triglyceride was measured with the Triglyceride GPO-PAP spectrophotometric assay (Roche Diagnostics), using Roche/Hitachi Modular Analyzer. Nonesterified fatty acid (NEFA) was measured with an enzymatic colorimetric method assay using the Wako NEFA-HR (2 (link)) reagent (Wako Chemical). High-density lipoprotein (HDL) cholesterol was measured by Roche WAKO Direct Homogenous assay. Very low-density lipoprotein cholesterol was calculated from the total HDL cholesterol measurements.
The 2H atom percentage excess in plasma glucose was determined using a Thermo “Voyager” single quadruple mass spectrometer with Thermo “Trace” gas chromatograph (Thermo Scientific). Plasma triglyceride was measured with the Triglyceride GPO-PAP spectrophotometric assay (Roche Diagnostics), using Roche/Hitachi Modular Analyzer. Nonesterified fatty acid (NEFA) was measured with an enzymatic colorimetric method assay using the Wako NEFA-HR (2 (link)) reagent (Wako Chemical). High-density lipoprotein (HDL) cholesterol was measured by Roche WAKO Direct Homogenous assay. Very low-density lipoprotein cholesterol was calculated from the total HDL cholesterol measurements.
10
Characterizing Garden Waste Decomposition
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The composite samples were retrieved from the serum bottles at predetermined intervals and subsequently centrifuged at 10,000 rpm for 5 min. The supernatant was filtered through a 0.45-μm membrane for the subsequent analysis of soluble chemical oxygen demand (sCOD) and filtered through a 0.22-μm membrane for the analysis of VFAs.
The COD, TS, and volatile solids (VS) were determined with standard methods (APHA, 2005 ). Acetate, propionate, butyrate, valerate, and caproate were measured by high-performance liquid chromatography (HPLC) (Bio-Rad, Hercules, California) using 5 mM H2SO4 as the mobile phase (Huang et al., 2022 (link)). The cumulative VFAs, calculated as acetate (g/L), were the sum of acetate, propionate, isobutyrate, and n-butyrate during fermentation. pH was measured with a pH meter (HACH, United States). The gas volume collected in a 1-L sampling bag was measured using an air pump every 1–3 days, and then, the gas was transferred back into the bag. The hydrogen content in both the gaseous samples in the sampling bag and the headspace was measured using gas chromatography apparatus equipped with a thermal conductivity detector (TCD) (Tianmei, GC7900, China). In addition, the decomposition of garden waste was assessed by Fourier-transform infrared (FT-IR) spectroscopy (Bruker; VERTEX 70; Germany) through a full scan across the wavenumber range of 400–4,000 cm−1.
The COD, TS, and volatile solids (VS) were determined with standard methods (APHA, 2005 ). Acetate, propionate, butyrate, valerate, and caproate were measured by high-performance liquid chromatography (HPLC) (Bio-Rad, Hercules, California) using 5 mM H2SO4 as the mobile phase (Huang et al., 2022 (link)). The cumulative VFAs, calculated as acetate (g/L), were the sum of acetate, propionate, isobutyrate, and n-butyrate during fermentation. pH was measured with a pH meter (HACH, United States). The gas volume collected in a 1-L sampling bag was measured using an air pump every 1–3 days, and then, the gas was transferred back into the bag. The hydrogen content in both the gaseous samples in the sampling bag and the headspace was measured using gas chromatography apparatus equipped with a thermal conductivity detector (TCD) (Tianmei, GC7900, China). In addition, the decomposition of garden waste was assessed by Fourier-transform infrared (FT-IR) spectroscopy (Bruker; VERTEX 70; Germany) through a full scan across the wavenumber range of 400–4,000 cm−1.
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