Vacuette z serum clot activator tubes

L-PRF was obtained from healthy volunteers (aged 25–37, n = 7, male and female) with written informed consent, and isolation was approved by the Medical Ethical Committee of the University Hospital of Leuven and Hasselt University (S58789/B322201628215/I/U). L-PRF was prepared according to the guidelines of the IntraSpin™ centrifuge (Intra-Lock International, Boca Raton, FL, USA). Briefly, 9 mL venous blood samples were collected in glass-coated tubes (VACUETTE^® Z Serum Clot Activator Tubes, Greiner Bio-One, Kremsmünster, Austria) and spun down immediately at 400 g (2700 rpm) for 12 min. The blood coagulates during this centrifugation step, and afterward the resulting L-PRF clots were collected and red blood cell remnants were removed (Figure 1A–D).

Complete blood count with leukocyte differential was performed from peripherally collected blood samples using Vacuette K3EDTA tubes and a Sysmex XT-2000iV hematology instrument using a preprogrammed monkey species profile (Sysmex America, NY). Plasma and serum were prepared by separation for 10 minutes at ambient temperature with centrifuge set to 1800 RCF. Serum chemistries were performed using the Piccolo Xpress Analyzer and General Chemistry 13 discs (Abaxis, Abbott Point of Care, NJ) from Vacuette Z Serum Clot Activator tubes (Greiner Bio-One, Monroe, NC).

Complete blood count with leukocyte differential was performed from peripherally collected blood samples using Vacuette K₃ ethylenediaminetetraacetic acid (EDTA) tubes and a Sysmex XT-2000iV hematology instrument using a preprogrammed monkey species profile (Sysmex America, NY). Plasma and serum were prepared by separation for 10 min at ambient temperature with centrifuge set to 1800 RCF. Serum chemistries were performed using the Piccolo Xpress Analyzer and General Chemistry 13 discs (Abaxis, Abbott Point of Care, NJ) from Vacuette Z Serum Clot Activator tubes (Greiner Bio-One, Monroe, NC).

Vacuette Z serum clot activator tubes (Greiner Bio-One, Monroe, NC, USA) were used for biochemical analyses. Becton Dickinson tubes (Trace Element, Serum, Franklin Lakes, NJ, USA) were used for zinc analyses. Polypropylene plastic syringes were purchased from BD (Hercules, CA, USA), and plastic tips and tubes (metal-free) were purchased from BioRad Laboratories (Hercules, CA, USA). Zinc sulfate heptahydrate (ZnSO₄·7H₂O) and Titrisol zinc standard were purchased from Merck (Darmstadt, Germany).

The rats were euthanized while under anesthesia with isoflurane (Isoba vet, Intervet, Schering-Plough Animal Health, Boxmeer, The Netherlands) mixed with nitrous oxide and oxygen after four-week intervention, after a 12-h fast with free access to tap water. Blood was drawn directly from the heart and collected in Vacuette Z Serum Clot Activator Tubes (Greiner Bio-one) for isolation of serum. Epididymal, renal and retroperitoneal white adipose tissues (WAT) were dissected out and weighed. Serum and WAT were snap-frozen in liquid nitrogen and stored at −80 °C until analysis.

All biochemical kits for alanine aminotransferase (ALT), aspartate amino transferases (AST), and alkaline phosphatase (ALP) were sourced from Beckman Coulter Inc. (California, USA). Terbinafine (Lamisil®; batch number U0638; marketed by Novartis Pharma Ltd., United Kingdom), itraconazole (Canditral®; batch number 01141282; marketed by Glenmark Pharmaceuticals Ltd., India), griseofulvin (Griseon®; batch number 178046; marketed by Plus Five Pharmaceuticals Ltd., Zimbabwe), fluconazole (Flumyc-200®; batch number AHP054014; marketed by Ipca Laboratories Ltd., India), and ketoconazole (Nizol®; batch number 13312; marketed by Intas Pharmaceuticals Ltd., India) were all sourced from a local pharmaceutical wholesaler. Standard diet pellets were obtained from National Foods Pvt. Ltd., Zimbabwe. Formaldehyde (37% solution), paraffin wax, haematoxylin, eosin, and other standard laboratory chemicals were sourced from Sigma-Aldrich (United Kingdom). Blood collection tubes (Vacuette® Z serum clot activator tubes) were sourced from Greiner Bio-One (United Kingdom). Microcentrifuge tubes (LW2075; batch number 110488) were sourced from Alpha Laboratories (Hampshire, United Kingdom).

Blood samples were collected on three different occasions on days 0, 15, and 29, using the cardiac puncture method. Day 0 was the day before initial drug administration. Blood samples collected from rats sacrificed on day 0 were for the determination of baseline liver enzyme levels in the clan of the rats. Day 15 was defined as the day after the rats had completed 14-day courses of drug treatment, that is, 24 hours after last dose. Day 29 was defined as the day after the rats had completed 28-day courses of treatment. Blood samples (4.0 ml) were collected using red top (black ring) vacutainers (6.0 ml Vacuette® Z serum clot activator tubes; Greiner Bio-One Ltd., UK) and were left to clot for 30 minutes before being spun down. Blood samples were spun down at 1200 rpm for 10 minutes and the serum transferred to 1.5 ml plastic microcentrifuge tubes (Alpha Laboratories, UK). The serum was then stored at −18°C up to the day of analysis (at most 7 days). The Beckman AU680® chemistry analyser was used to determine plasma levels of ALP, AST, and ALT. Aspartate transaminase (AST) and alanine transaminase (ALT) activity in serum were assayed using a procedure based on the method developed by Wróblewski and Ladue [27 (link)–29 ]. Alkaline phosphatase (ALP) activity in serum was assayed by a procedure based on the method developed by Bowers Jr. and McComb [30 (link)].