Sultan Qaboos University Med J, August 2013, Vol. 13, Iss. 3, pp. 411-416, Epub. 25th Jun 13 Submitted 11TH Sep 12 Revisions Reqd. 25TH Nov 12 & 5TH Feb 13; Revisions Recd. 2ND Jan & 10TH Feb 13 Accepted 17TH Mar 13 1Department of Paediatrics, College of Medicine & Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates; 2Tawam Hospital, Al Ain, United Arab Emirates; 3Faculty of Medicine & Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates *Corresponding Author e-mail: aljasmif@uaeu.ac.ae استهالك األوكسجني ىف املايتوكوندريا من قبل القلفة و اخلاليا الليفية فاطمة اجل�شمي، ثكالت برامازان ، عدنان �شويد، بهجت �شحاري، هاريف بنيفي�شكي، عبد القادر �شويد امللخ�ص: الهدف: درا�شة اإمكانية ا�شتخدام طريقة ا�شتهالك االأوك�شجني يف امليتوكوندريا لقيا�س التنف�س اخللوي يف عينات القلفة و اخلاليا الليفية با�شتخدام جهاز حتليل االأوك�شجني احل�شا�س للفو�شفور. الطريقة: مت جمع عينات من القلفة لر�شع طبيعيني على الفور بعد اخلتان وقيا�س )II( د ب بورفريين بنزو ترتا ميزو مادة با�شتخدام االأوك�شجني ا�شتهالك �رسعة حتديد مت اخللوي. التنف�س لقيا�س وا�شتخدمت حمكمة زجاجات يف الزمن مع خطية بطريقة االأوك�شجني تركيز تناق�س النتائج: امليتوكوندريا. يف للفو�شفور احل�شا�س حتللها �رسعة االإغالق حتتوي على القلفة وجلوكوز مما يثبت ا�شتهالك االأوك�شجني بوا�شطة ال�شيتوكروم اوك�شيداز اخللوي. مادة ال�شيانيد اأوقفت ا�شتهالك القلفة تنف�س �رسعة متو�شط كان االأوك�شجني. ا�شتخدام عن امل�شئولة هي امليتوكندريا اأن توؤكد النتائج هذه القلفة. قبل من االأوك�شجني 0.02 + 0.074 دقيقة 2اأ مرت -1 جمم -1 )العدد=23( و�رسعة تنف�س اخلاليا اللليفية دقيقة 2اأ مرت 2.43 + 9.84 -1 جمم -1 )العدد=15(. كان تنف�س اخلاليا الليفية اأقل يف املر�شى امل�شابني بنق�س الثنائي هيدروليباميد )DLD( .هذا النق�س حت�شن باإ�شافة الثيامني اأو الكارنيتني. اخلال�سه: ميكن ا�شتخدام القلفة واخلاليا الليفية لتقييم التنف�س اخللوي. الفائدة ال�رسيرية من عينات القلفة للك�شف عن ا�شطرابات الطاقة احليوية يتطلب مزيداً من البحث. مفتاح الكلمات: االأوك�شجني؛ امليتوكوندريا؛ القلفة؛ التنف�س؛ اخلاليا الليفية؛ نق�س الثنائي هيدروليباميد؛ الثيامني؛ الكارنيتني. abstract: Objectives: This study investigated the feasibility of using a phosphorescence oxygen analyser to measure cellular respiration (mitochondrial O2 consumption) in foreskin samples and their fibroblast-rich cultures. Methods: Foreskin specimens from normal infants were collected immediately after circumcision and processed for measuring cellular respiration and for culture. Cellular mitochondrial O2 consumption was determined as a function of time from the phosphorescence decay of the Pd (II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. Results: In sealed vials containing a foreskin specimen and glucose, O2 concentration decreased linearly with time, confirming the zero-order kinetics of O2 consumption by cytochrome oxidase. Cyanide inhibited O2 consumption, confirming that the oxidation occurred mainly in the mitochondrial respiratory chain. The rate of foreskin respiration (mean ± SD) was 0.074 ± 0.02 μM O2 min -1 mg-1 (n = 23). The corresponding rate for fibroblast-rich cultures was 9.84 ± 2.43 μM O2 min -1 per 107 cells (n = 15). Fibroblast respiration was significantly lower in a male infant with dihydrolipoamide dehydrogenase gene mutations, but normalised with the addition of thiamine or carnitine. Conclusion: The foreskin and its fibroblast-rich culture are suitable for assessment of cellular respiration. However, the clinical utility of foreskin specimens to detect disorders of impaired cellular bioenergetics requires further investigation. Keywords: Oxygen; Mitochondria; Foreskin; Respiration; Fibroblasts; Dihydrolipoamide dehydrogenase; Thiamine; Carnitine. Mitochondrial Oxygen Consumption by the Foreskin and its Fibroblast-rich Culture *Fatma Al-Jasmi,1 Thachillath Pramathan,1 Adnan Swid,2 Bahjat Sahari,2 Harvey S. Penefsky,3 Abdul-Kader Souid1 clinical & basic research Advances in Knowledge - This study demonstrates the feasibility of using foreskin samples to measure cellular respiration (cellular mitochondrial oxygen consumption). Application to Patient Care - Foreskin specimens and their fibroblast-rich cultures can be used to screen for disorders of impaired cellular bioenergetics. - This study shows the potential use of fibroblast respiration to predict responses to therapeutic interventions. Mitochondrial Oxygen Consumption by the Foreskin and its Fibroblast-rich Culture 412 | SQU Medical Journal, August 2013, Volume 13, Issue 3 Clinicians frequently use skin and muscle biopsies for investigating mitochondrial disorders.1–4 The skin is also used for generating fibroblasts, which are easily obtained and used for repetitive biochemical analyses and research purposes. Circulating lymphocytes are also available and suitable for these measurements.4–7 More novel approaches for monitoring cellular bioenergetics have been reported recently.8–10 Al-Jasmi et al. described the use of a phosphorescence oxygen analyser to measure lymphocyte respiration in patients.10 Their method was based on previously published principles.11 The lymphocytes from patients were shown to be suitable for the screening of certain mitochondrial disorders. The same analytical tool is applied here to measure respiration in foreskin samples and their fibroblast-rich cultures. The results demonstrate that the foreskin tissue permits accurate determination of cellular mitochondrial O2 consumption. The primary aim of this study was to investigate the use of the phosphorescence oxygen analyser to measure cellular respiration (mitochondrial O2 consumption) in foreskin specimens and their fibroblast-rich cultures. The secondary aim was to utilise the foreskin to screen for metabolic disorders that impair cellular respiration (mitochondrial O2 consumption and accompanying adenosine triphosphate [ATP] synthesis). The hypothesis was that the foreskin and its fibroblast-rich culture can be utilised for assessment of cellular metabolic fuels and their energy conversion processes. Methods The following reagents and solutions were used. A Pd (II) complex of meso-tetra-(4-sulfonatophenyl)- tetrabenzoporphyrin was obtained from Porphyrin Products (Logan, Utah, USA). Minimum essential medium (MEM Alpha Modification), phosphate- buffered saline (PBS), fetal bovine serum, trypsin, penicillin, streptomycin and lyophilised collagenase (clostridiopeptidase prepared from Clostridium histolyticum, Cat. No. 17018-029) were obtained from Invitrogen Corporation (Carlsbad, California, USA). The thiamin HCl injection (100 mg/mL, 300 mM, m.w. 337.3) was obtained from APP Pharmaceuticals (Division of Fresenius Kabi, Schaumburg, Illinois, USA). The levocarnitine injection (1.0 g/5 mL, 1.24 M, m.w. 161.2), glucose oxidase (powder from Aspergillus niger), D(+) glucose anhydrous and remaining reagents were purchased from Sigma-Tau Pharmaceuticals, Inc. (Gaithersburg, Maryland, USA). Two mg of the Pd phosphor were dissolved in 1.0 mL of distilled water (dH2O) and stored at -20o C. A glucose oxidase solution was prepared in dH2O (10 mg/mL) and stored at -20 o C. One M of sodium cyanide (NaCN) was prepared in dH2O; the pH was adjusted to ~7.0 with 12N HCl and stored at -20o C. PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.4 mM KH2PO4, pH 7.4) containing 10 mM glucose was stored at 4o C. Foreskin specimens (22–34 mg) were collected from normal infants (<6 months of age) immediately after circumcision and stored at 4o C in 50 mL MEM supplemented with penicillin and streptomycin for <24 hours until O2 analysis or culture. For O2 measurement, the foreskin samples were placed in 1-mL O2 vials and processed as described below. For culture, the foreskin specimens were incubated at 37o C in a sterile vial containing PBS with 2.0 mg/mL collagenase. After tissue disintegration (typically in 2–3 hours), the samples were transferred to 25-cm3 tissue culture flasks containing 10 mL MEM with fetal-bovine serum, penicillin, and streptomycin. For passage, the cells were washed with 5 mL PBS and then treated at 37o C with 2 mL of 0.25% trypsin (w/v in PBS) for 5 minutes. The flasks were inspected for cell detachment; 1.0 mL of fetal-bovine serum and 5.0 mL of MEM with fetal-bovine serum, penicillin and streptomycin were then added. The suspension was split between 2–4 flasks, depending on the cell concentration. Fibroblast-rich cultures were harvested at confluence. Cells were washed with PBS and treated at 37o C with 0.25% trypsin for 5 minutes. The cells were then collected in MEM with fetal-bovine serum, penicillin and streptomycin and centrifuged at 1,000 g (25o C for 5 minutes). The pellet was suspended in 1.3 mL of Pd solution (PBS, 3 µM Pd phosphor, 0.5% fat-free albumin, and 5 mM glucose) and processed for O2 measurement. Thiamin or carnitine was added to confluent fibroblast-rich cultures and the cells were harvested after 24 hours. Cell count and viability were determined by light microscopy using a haemocytometer under standard trypan blue staining condition. Only trypan blue-negative cells (>95%) were counted. It Fatma Al-Jasmi, Thachillath Pramathan, Adnan Swid, Bahjat Sahari, Harvey S. Penefsky and Abdul-Kader Souid Clinical and Basic Research | 413 is worth emphasising that foreskin samples should be immediately placed in a large volume (e.g., 50 mL) of ice-cold medium and stored at 4o C until processing. This procedure produces better sample viability. Sample collection from all participants was approved by the institutional review board for protection of human subjects. Informed consent was obtained for each patient. Ethical permission was granted by the Al Ain Medical District Human Research Ethics Committee (16th April 2012, Protocol No. 11/59). The rate of cellular respiration was determined using a phosphorescence analyser to measure the concentration of dissolved oxygen as a function of time.10 This method is based on the principle that O2 quenches the phosphorescence of a palladium phosphor.11 The Pd (II) derivative of meso-tetra- (4-sulfonatophenyl)-tetrabenzoporphyrin had a maximum absorption at 625 nm and a maximum phosphorescence emission at 800 nm. Samples were exposed to light flashes (10 per second) from a pulsed light-emitting diode array with a peak output of 625 nm. Emitted phosphorescent light was detected by a Hamamatsu photomultiplier tube (Hamamatsu, Japan) after first passing it through a wide-band interference filter centered at 800 nm. Amplified phosphorescence was digitised at 1–2 MHz using an analogue/digital converter (PCI-DAS 4020/12 I/O Board) with 1 to 20 MHz outputs. Pulses were captured at 1.0 MHz. The phosphorescence decay rate (1/τ) was characterised by a single exponential I = Ae- t/τ, where I = Pd phosphor phosphorescence intensity. The values of 1/τ were linear with dissolved O2: 1/ τ = 1/τº + kq[O2], where 1/τ = the phosphorescence decay rate in the presence of O2, 1/τº = the phosphorescence decay rate in the absence of O2 and kq = the second-order O2 quenching rate constant in sec-1 µM-1.11 Cellular respiration was measured at 37o C in 1-mL sealed vials containing PBS, 3 µM Pd phosphor, Table 1: Respiration of foreskin samples and their fibroblast-rich cultures from normal infants Rates of Respiration tissue Foreskin* (µM O2 min -1 mg-1) Fibroblast† (µM O2 min -1 per 107 cells) Lymphocyte‡ (µM O2 min -1 per 107 cells) Mean ± SD (n) 0.074 ± 0.02 (23) 9.84 ± 2.43 (15) 2.0 ± 0.9 (20) Median (range) 0.074 (0.04–0.13) 10.50 (6.6 –14.3) 2.0 (0.9–3.7) SD = standard deviation; n = number of subjects. * The samples were stored at 4o C in MEM for <24 hours before O2 measurement and culture. † The fibroblasts were prepared from foreskin samples; 107 fibroblasts had a net weight of 70.4 ± 12.5 mg (n = 13). ‡ The data are from reference 10. Table 2: Respiration of foreskin samples, fibroblasts and lymphocytes from patients Gender Age Clinical presentation Rates of respiration Foreskin Fibroblast value ± SD lymphocyte M 1 yr DLD homozygous gene mutation (c.1436A>T) Reduced PDHc activity in fibroblasts Not done 5.7 ± 1.4 (n = 6)* P <0.0001 0.79‡ P = 0.091 M 2 m Congenital lactic acidosis 0.076 5.6 ± 0.3 (n = 3)† P = 0.002 1.0 F 15 m Global developmental delay Not done 10.1 ± 0.4 (n = 2)‡ 0.62 DLD = dihydrolipoamide dehydrogenase; PDHc = pyruvate dehydrogenase complex; SD = standard deviation; n = number of repeats using independence samples; P = P value. The lymphocyte and fibroblast respiration is expressed in μM O2 min -1 per 107 cells. The foreskin respiration is expressed in μM O2 min -1 per mg. The results in Table 2 are in comparison to Table 1. *The fibroblasts were from a skin biopsy. The P value is for fibroblast respiration in normal infants versus fibroblast respiration in the patient. †The fibroblasts were from a foreskin sample. ‡The patient is receiving thiamin. This rate was 1.5 μM O2 per min per 10 7 cells at 1 week of age (from reference 10). The P-value is for lymphocyte respiration in age-matched normal individuals versus lymphocyte respiration in the patient.4 Mitochondrial Oxygen Consumption by the Foreskin and its Fibroblast-rich Culture 414 | SQU Medical Journal, August 2013, Volume 13, Issue 3 0.5% fat-free albumin, and 5 mM glucose. The respiratory substrates were endogenous metabolic fuels supplemented by glucose. O2 concentration (calculated using the equation 1/ τ = 1/τº + kq[O2]) decreased linearly with time, indicating the kinetics of mitochondrial O2 consumption was zero-order. The rate of respiration (k, in µM O2/min) was, thus, the negative of the slope d[O2]/dt. NaCN inhibited respiration by at least 96%, confirming oxygen was mainly consumed by the mitochondrial respiratory chain. A programme was developed using Microsoft Visual Basic 6 and Access Database 2007 (Microsoft Corp., Redmond, Washington, USA), and Universal Library (Measurement Computing Corporation, Norton, Massachusetts, USA) components.12,13 Calibration with β-D-glucose plus glucose oxidase was performed as follows. Glucose oxidase catalyses the oxidation reaction of β-D-glucose + O2 to D-glucono-1,5-lactone + H2O2. The reactions contained PBS + 3 µM Pd phosphor, 0.5% fat-free albumin, 50 µg/mL glucose oxidase, and 0-500 µM β-glucose. The value of kq, the negatives of the slope of 1/τ versus [β -glucose], was 101.1 sec-1 µM-1. Data were analysed using Statistical Package for the Social Sciences (SPSS), Version 19 (IBM Corp., Chigaco, Illinois, USA). The Mann-Whitney nonparametric test (2 independent variables) was used to compare treated and untreated samples. Results The rates of foreskin and fibroblast respiration are shown in Table 1, and representative runs are shown in Figure 1A. A summary of all studied samples is shown in Figure 1B and Table 1. For comparison, the reference values for lymphocyte respiration are also shown in Table 1.10 The rate of respiration (kc, µM O2 min -1 mg-1) in foreskin samples that was determined within one hour of circumcision was 0.076 ± 0.01 (coefficient of variation [Cv] = 14%; n = 8). For samples that were stored at 4o C in MEM for 16 to 23 hours, the respiration rate was 0.074 ± 0.025 (Cv = 33%, n = 16, P = 0.697), and for samples that were stored at 4o C in MEM for >24 hours the rate was 0.033 ± 0.023 (Cv = 70%, n = 3, P = 0.012). Thus, the samples were stable for up to 23 hours. An infant with congenital lactic acidaemia (plasma lactate 18 mM; normal range <1.5) was investigated. His plasma lactate decreased to 6–8 mM within one week of thiamin treatment (500 mg/ Kg). The pyruvate dehydrogenase complex (PDHc) activity in fibroblasts was low (2.7 mU/UCS; controls = 9.7 to 36 mU/UCS; this test was done on a clinical sample analysed by Stichting Klinisch-Genetisch Centrum, Nijmegen, Netherlands. Sequencing the dihydrolipoamide dehydrogenase (DLD) gene showed homozygous c.1436A> T (p. Asp 479 Val) (Refseq accession number NM_000108). The respiration rate of the infant’s lymphocytes and fibroblasts was consistently low [Table 2]. His fibroblasts were then treated with various concentrations of thiamin (cofactor of PDHc) and carnitine over 24 hours, as patients with impaired pyruvate dehydrogenase complex are typically treated with these agents. The value of kc (µM O2 min-1 mg-1) with 100 µM thiamin was 4.3, with 200 µM thiamin 7.1, and with 400 µM thiamin 8.6. The value of kc with 50 µM carnitine was 5.1, with 100 µM carnitine 8.6, and with 200 µM carnitine 8.8. Another patient with congenital lactic acidosis, a 2-month-old infant, was also investigated. His plasma lactate was 17.9 mM and cerebrospinal fluid lactate 8.27 mM (normal = 0.86–2.19). His foreskin respiration (0.076 µM O2 min -1 per mg) and lymphocyte respiration (1.0 µM O2 min -1 per 107 cells) was normal, while his fibroblast (from a foreskin sample) respiration was low (5.6 ± 0.3 µM O2 min -1 per 107 cells, n = 3, P = 0.002). A 15-month-old female presented with global developmental delay and failure to thrive. The urine organic acid analysis showed elevated 2-ketoglutaric acid and lactate levels. Plasma alanine was mildly elevated. The lymphocyte respiration was low (0.62 µM O2 min -1 per 107 cells) while the fibroblast respiration was normal (10.1 ± 0.4 µM O2 min -1 per 107 cells, n = 2). Discussion The main finding in this study is the consistency of the rate of cellular respiration in foreskin samples (Cv = 27%) and their fibroblast-rich cultures (Cv = 25%) [Table 1]. These variations are significantly lower than that of the lymphocytes (Cv = 45%) [Table 1], which are typically more fragile than the ectodermal tissue. Therefore, the foreskin appears to be a reliable source of cells for measuring cellular respiration. The other important observation is the relative stability of foreskin samples over several hours. These features make the foreskin suitable for Fatma Al-Jasmi, Thachillath Pramathan, Adnan Swid, Bahjat Sahari, Harvey S. Penefsky and Abdul-Kader Souid Clinical and Basic Research | 415 metabolic disorder screening. The term cellular bioenergetics covers all of the biochemical processes involved in the energy conversion. Cellular respiration (mitochondrial oxygen consumption), on the other hand, implies the generation of metabolic fuels, delivery of metabolites and O2 to the mitochondria, oxidation of reduced metabolic fuels with passage of electrons to O2, and synthesis of ATP. Thus, impaired cellular bioenergetics entails an interference with any of these critical processes. To our knowledge, this report is the first to show the feasibility of measuring cellular respiration in the foreskin. The procedure is relatively simple, and the tissue is stable at 4o C for several hours after circumcision. The clinical applications of using foreskin samples include the immediate reading of cellular respiration, as well as processing the tissue for fibroblast-rich cultures, with a success rate of >90%. The rates of oxygen consumption by fibroblasts and lymphocytes differ markedly (P <0.001); the lymphocyte rate is about 5 times lower than that of the fibroblast [Table 1]. Due to their in vitro stability, fibroblasts appear to be more reliable for measuring respiration than circulating lymphocytes. The infant with the homozygous DLD gene mutations (c.1436A>T), and reduced PDHc activity, had low rates of fibroblast and lymphocyte respiration. These results confirm the suitability of using these types of tissues in the screening for this disorder. Thiamine and carnitine supplements are recommended for patients with impaired pyruvate dehydrogenase complexes. Therefore, these agents were tested on the PDH-deficient patient. Fibroblast respiration normalised with the addition of thiamin or carnitine (see Results section). Thus, the potential response to these therapeutic interventions could be predicted in vitro. In one patient with congenital lactic acidosis, the foreskin and lymphocyte respiration rates were normal, while the fibroblast respiration was low.The 15-month-old female with global developmental delay of undetermined aetiology had low lymphocyte respiration, but normal fibroblast respiration [Table 2]. The source of these discrepancies is unclear, but could reflect heteroplasmy;3 thus, respiration should always be determined in multiple tissue sources. In one study, 50% of patients with confirmed respiratory chain defects had abnormal measurements in muscle and lymphocyte samples, 45% in muscle samples only and 5% in lymphocyte samples only.4 Pearson’s syndrome, on the other hand, consistently expresses abnormalities in the Figure 1 Panels A & B: Rates of cellular respiration of foreskins and foreskin cultures from healthy infants. Panel A: Representative runs of a foreskin sample (33 mg) and its fibroblast-rich culture (1.2 x 107 cells) from the same infant. The O2 measurements were performed at 37º C in 1-mL sealed vials of PBS supplemented with 5 mM glucose, 3 µM Pd phosphor and 0.5% fat-free bovine serum albumin. The rates of respiration (k, in µM O2 min -1) were set as the negative of the slopes of [O2] versus time. The slopes were calculated from the best-fit curve (R 2 >0.920). The values of kc in µM O2 min-1 per mg (foreskin) and µM O2 min -1 per 107 cells (fibroblast-rich culture) are also shown. The additions of 5 mM NaCN and 50 μg/mL glucose oxidase are shown. Glucose oxidase (catalyses the reaction of D-glucose + O2 to D-glucono- δ-lactone + H2O2) depleted the remaining O2 in the solution. The depletion of O2 after the addition of glucose oxidase confirmed that the halt of respiration following the cyanide injection occurred despite available O2 in the solution. Panel B: The values of kc for all studied foreskins (white circles) and foreskin cultures (black circles) are shown. The short horizontal lines on the y-axis reflect the mean values. Mitochondrial Oxygen Consumption by the Foreskin and its Fibroblast-rich Culture 416 | SQU Medical Journal, August 2013, Volume 13, Issue 3 lymphocytes.5 These results again highlight the need for investigating several tissues. Cellular respiration is a reliable indicator of mitochondrial function [Figure 1]. This important biomarker is underutilised, mostly due to the traditional limitations of the polarographic method.14 Polarography (Clark-type O2 electrode) and spectroscopy have been used as analytical methodologies for measuring O2 consumption by fresh lymphocytes.14 Reported rates of lymphocyte respiration (all in nmol O2 min -1 per 107 cells) include 3.5 ± 0.5, 2.0 ± 0.07 (varied by the cell density), and 1.0 ± 0.2 (in equine lymphocytes).5–7 More recently, the fluorescence and phosphorescence O2 sensors have permitted relatively simple and accurate monitoring of respiration in clinical samples of small quantities.8–11 Measurements of mitochondrial respiration in digitonin-permeabilised fibroblasts and in isolated mitochondria from muscle specimens using phosphorescent microplates have been reported.8 Other analytic instruments for assessing cellular bioenergetics are also described.9 Of note, the phosphorescence oxygen analyser used here was calibrated using the Clark electrode, and validated for measurements of respiration in various cells and tissues.10,11,15 Conclusion This study demonstrates the feasibility of using the foreskin and its fibroblast-rich culture to measure cellular mitochondrial O2 consumption. This tissue is dispensable and relatively stable for several hours; it is therefore ideal for screening. Muscle and skin biopsies, on the other hand, are relatively invasive and available in minute quantities. These more precious samples are thus more suitable for confirmatory biochemical or molecular testing. The clinical utility of foreskin samples in detecting disorders of impaired cellular bioenergetics, however, requires further investigation. References 1. Chretien D, Rustin P. Mitochondrial oxidative phosphorylation: pitfalls and tips in measuring and interpreting enzyme activities. J Inherit Metab Dis 2003; 26:189–98. 2. Chretien D, Rustin P, Bourgeron T, Rotig A, Saudubray JM, Munnich A. Reference charts for respiratory chain activities in human tissues. Clin Chim Acta 1994; 228:53–70. 3. Rustin P, Chretien D, Bourgeron T, Gerard B, Rotig A, Saudubray JM, et al. Biochemical and molecular investigations in respiratory chain deficiencies. Clin Chim Acta 1994; 228:35–51. 4. Robinson BH. Use of fibroblast and lymphoblast cultures for detection of respiratory chain defects. Methods Enzymol 1996; 264:454–64. 5. Rotig A, Cormier V, Blanche S, Bonnefont JP, Ledeist F, Romero N, et al. Pearson's marrow-pancreas syndrome. A multisystem mitochondrial disorder in infancy. J Clin Invest 1990; 86:1601–8. 6. Hedeskov CJ, Esmann V. Respiration and glycolysis of normal human lymphocytes. Blood 1966; 28:163– 74. 7. Pachman LM. The carbohydrate metabolism and respiration of isolated small lymphocytes. In vitro studies of normal and phytohemagglutinin stimulated cells. Blood 1967; 30:691–706. 8. Jonckheere AI, Huigsloot M, Janssen AJ, Kappen AJ, Smeitink JA, Rodenburg RJ. High-throughput assay to measure oxygen consumption in digitonin- permeabilized cells of patients with mitochondrial disorders. Clin Chem 2010; 56:424–31. 9. Ferrick DA, Neilson A, Beeson C. Advances in measuring cellular bioenergetics using extracellular flux. Drug Discov Today 2008; 13:268–74. 10. Al-Jasmi F, Penefsky HS, Souid AK. The phosphorescence oxygen analyzer as a screening tool for disorders with impaired lymphocyte bioenergetics. Mol Genet Metab 2003; 104:529–36. 11. Lo LW, Koch CJ, Wilson DF. Calibration of oxygen- dependent quenching of the phosphorescence of Pd- meso-tetra (4-carboxyphenyl) porphine: a phosphor with general application for measuring oxygen concentration in biological systems. Anal Biochem 1996; 236:153–60. 12. Anderson NR, Lee ES, Brockenbrough JS, Minie ME, Fuller S, Brinkley J, et al. Issues in biomedical research data management and analysis: needs and barriers. J Am Med Inform Assoc 2007; 14:478–88. 13. Shaban S, Marzouqi F, Al Mansouri A, Penefsky HS, Souid AK. Oxygen measurements via phosphorescence. Comput Methods Programs Biomed 2010; 100:265–8. 14. Clark LC. Electrochemical device for chemical analysis. US patent no. 291338, 1959. 15. Al-Jasmi F, Al-Suwaidi AR, Al-Shamsi M, Marzouqi F, Al Mansouri, Shaban S, et al. Phosphorescence oxygen analyzer as a measuring tool for cellular bioenergetics. From: http://cdn.intechopen.com/ pdfs/30413/InTech-Phosphorescence_oxygen_ a n a l y z e r _ a s _ a _ m e a s u r i n g _ to o l _ fo r _ cel l u l a r _ bioenergetics.pdf Accessed: Aug 2012.