Fatma A. Al-Jasmi*, Thachillath Pramathan, Hager Kowash and Abdul-Kader Souid
Department of Pediatrics, United Arab Emirates University, College of Medicine and Health Sciences, P.O. Box 17666, Al Ain, United Arab Emirates
Received date: March 11, 2016; Accepted date: May 05, 2016; Published date: May 10, 2016
Citation: Al-Jasmi F, Pramathan T, Kowash H, Souid AK (2016) In Vitro Study on the Response of Fibroblast Cellular Respiration to Lipoic acid, Thiamine and Carnitine in Patients with Dihydrolipoyl Dehydrogenase Deficiency. J Mol Genet Med 10:214. doi:10.4172/1747-0862.1000214
Copyright: © 2016 Al-Jasmi FA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Objectives: This study examined in vitro responses of fibroblast cellular respiration to lipoic acid, thiamine and carnitine in patients with dihydrolipoyl dehydrogenase (DLD) deficiency. This disorder impairs cellular bioenergetics and these compounds are used to improve clinical manifestations of the disease. The study aimed to utilize mitochondrial O2 consumption as a surrogate biomarker for examining cellular responses to metabolic therapies.
Methods: Cultured fibroblasts from three patients were treated with therapeutic concentrations of the compounds for 24 hours. Cells were then harvested and processed for measuring respiration using phosphorescence oxygen analyzer. Patients 1 and 2 were severely symptomatic infants with homozygous c.1436A>T mutation in the DLD gene. Patient-3 was a mildly symptomatic adolescent with homozygous c.685G>T mutation.
Results: The rate of respiration (mean ± SD, n=6, μM O2 min-1/107 cells) in fibroblasts from a normal infant was 9.3 ± 1.6, in fibroblasts from Patient-1 was 5.1 ± 0.9 (p=0.001), in fibroblasts from Patient-2 was 7.4 ± 1.4 (p=0.051), and in fibroblasts from Patient-3 was 10.3 ± 3.3 (p=0.836). In normal fibroblasts, respiration decreased by the thiamine (p=0.012) and carnitine (p=0.023) treatments. In Patient-1, respiration increased by the lipoic acid (p<0.002), thiamine (p<0.001), and carnitine (p=0.018) treatments; this patient clinically responded to thiamine. In Patient-2, respiration decreased by the thiamine (p=0.026) and carnitine (p=0.008) treatments; this patient did not respond to these drugs. In Patient-3, respiration increased by the carnitine (p=0.012) treatment; the patient clinically responded to carnitine.
Conclusions: The results show cellular respiration is a suitable biomarker for the disease. The significance of using this tool to assess responses to therapies requires further studies.
ATP: Adenosine Triphosphate; Coa: Coenzyme A; CO2: Carbon Dioxide; DLD: Dihydrolipoyl Dehydrogenase; dH2O: Distilled Water; MEM: Minimum Essential Medium; NAD: Nicotinamide Adenine Dinucleotide; O2: Oxygen; Pd: Palladium; PDHc: Pyruvate Dehydrogenase Complex; SD: Standard Deviation
We recently reported on the use of phosphorescence oxygen analyzer to measure cellular respiration in fibroblasts, lymphocytes and foreskins from patients with inborn error of metabolism [1-3]. The same method is employed here to study fibroblast cellular respiration (mitochondrial O2 consumption) in patients with dihydrolipoyl dehydrogenase (DLD) deficiency (MIM #238331). This autosomal recessive disorder results mostly from mutations in the DLD gene. Patients present with and early-onset lactic acidosis and developmental delay or late-onset neurological and hepatic dysfunctions . These patients are routinely treated with combinations of DL-lipoic acid, thiamin and carnitine [5-7].
Lipomide dehydrogenase (EC 18.104.22.168; dihydrolipoyl dehydrogenase or E3 component) is a flavoprotein enzyme that oxidizes dihydroliopamide to lipoamide. E3 is an essential component of three related mitochondrial enzyme complexes involved in energy biotransformations, pyruvate dehydrogenase, α-ketoglutarate (or 2- oxoglutarate) dehydrogenase, and branched-chain α-keto acid dehydrogenase. Deficiency of E3, thus, is expected to impair cellular bioenergetics.
The pyruvate dehydrogenase complex couples glycolysis to the citric acid cycle; it catalyzes the pyruvate decarboxylation reaction that generates acetyl-CoA. The enzyme has three components, pyruvate dehydrogenase (EC 22.214.171.124, or E1 component; thiamine pyrophosphate is a co-enzyme of E1), dihydrolipoyl transacetylase (EC 126.96.36.199, or E2 component; lipoic acid is a co-enzyme of E2), and the E3 component (flavin adenine dinucleotide is a co-enzyme of E3).
The α-ketoglutarate dehydrogenase complex catalyzes a rate limiting reaction in the citric acid cycle (α-ketoglutarate + NAD+ + CoA → succinyl CoA + CO2 + NADH). The enzyme has three components, α-ketoglutarate dehydrogenase (EC 188.8.131.52, or E1 component; thiamine pyrophosphate is a co-enzyme of E1), dihydrolipoyl succinyltransferase (EC 184.108.40.206, or E2 component; lipoic acid is a co-enzyme of E2), and the E3 component.
The branched-chain α-keto acid dehydrogenase complex catalyzes oxidative decarboxylations of L-leucine, L-isoleucine, L-valine, and their derivatives (including pyruvate). The enzyme has three components, α-ketoacid dehydrogenase (E1 component; thiamine pyrophosphate is a co-enzyme of E1), dihydrolipoyl transacylase (E2 component; lipoic acid is a co-enzyme of E2), and the E3 component .
Thus, the phosphate derivative of thiamine (thiamine diphosphate, also known as thiamine pyrophosphate) and the lipoyl moiety are essential catalytic co-factors in these critical energy-generating metabolic reactions. L-carnitine, on the other hand, is required for transport of fatty acids from the cytosol to the mitochondria as a source of energy. Therefore, known functions of the three compounds justify their administration to patients with lipomide dehydrogenase deficiency.
This study investigated the in vitro responses of fibroblast cellular respiration to lipoic acid, thiamine, and carnitine in patients with DLD deficiency. Our hypothesis was that these cofactors support cellular bioenergetics and improve respiration in patients with DLD deficiency.
Pd (II) complex of meso-tetra-(4-sulfonatophenyl)- tetrabenzoporphyrin was purchased from Porphyrin Products (Logan, UT, USA). Pd phosphor solution (2.0 mg/ml = 2 mM) was prepared in dH2O and stored at −20°C. Minimum Essential Medium (MEM Alpha Modification, #11900-016) was purchased from Gibco (Life Technology Corporation, Paisley, UK). Thiamine HCl Injection (100 mg/mL, 296.5 mM, m.w. 337.3) was purchased from APP Pharmaceuticals (Schaumburg, IL, USA). Levocarnitine Injection (200 mg/mL, 1.24 M, m.w. 161.2) and (±)-α-lipoic acid (m.w. 206.33) were purchased from Sigma-Tau Pharmaceuticals. The three compounds were stored at 4°C; appropriate dilutions were made in MEM and used immediately.
Tissue collection from all participants was approved by the institutional ethical review board for protection of human subjects. Informed consent was obtained for each patient. The fibroblast cultures were prepared from foreskin specimen of a normal infant and skin biopsies of three patients and processed as previously described .
Cells were treated at confluence with lipoic acid (50 or 100 μM), thiamine (233, 466, or 932 μM), or carnitine (100, 200, or 400 μM). Cells were harvested 24 hours after treatment and processed for measuring cellular respiration as previously described [1-3]. The drugs were used at saturating concentrations and there were no significant concentration-dependent effects. Therefore, the results for each compound were grouped.
Data were analyzed using SPSS statistical package (version 20). The nonparametric test (2 independent variables; Mann-Whitney) was used to compare treated and untreated samples.
The rate of cellular respiration (mean ± SD, in μM O2 min-1 per 107 cells) in fibroblasts (passage #3) from a normal infant was 9.3 ± 1.6 (n=7). The corresponding rate in lipoic acid-treated fibroblasts was 8.0 ± 2.5 (n=8, p =0.336), thiamine-treated fibroblasts was 7.0 ± 1.1 (n=9, p =0.012), and carnitine-treated fibroblasts was 7.1 ± 2.1 (n=9, p =0.023), Figures 1A and 1B.
Figure 1: Cellular respiration in fibroblasts from a normal infant. Fibroblasts were treated at confluence with no addition or with the addition of lipoic acid, thiamine, or carnitine. The cells were harvested 24 hours after treatment and processed for measuring cellular respiration. The rate of respiration (k , μM O2 min-1) was the negative of the slope of [O2] vs. t. (A) Representative O2 measurements from seven to nine separate experiments for each condition are shown. The lines are best linear fit. The values of kc (μM O2 min-1 per 107 cells) are shown at the bottom of each run. (B) Summary of all measurements is shown. The concentrations of lipoic acid were 50 and 100 μM; thiamine 233, 466 and 932 μM; and carnitine 100, 200 and 400 μM. The results for each compound were grouped since there were no significant concentration-dependent effects. The horizontal lines are mean.
Feasible explanation of the lower rate of respiration with cofactors is more efficient oxidation of reduced metabolic fuels in the mitochondrial respiratory chain.
This infant presented at 24 hours of age with lactic acidosis (Table 1). Pyruvate dehydrogenase complex (PDHc) activity in his fibroblasts was severely reduced. Sequencing the DLD gene showed the homozygous c.1436A>T (p.Asp479Val) mutation. He was treated with thiamine (700 mg/day) and carnitine (100 mg/kg). Cellular respiration was measured in fibroblasts prepared from passage #3. Representative measurements of his fibroblast respiration in the presence of designated concentrations of lipoic acid, thiamine, and carnitine are shown in Figure 2A; a summary of all results is shown in Figure 2B.
|Patient 1||Patient 2||Patient 3|
|Age at presentation||Birth||Birth||One year|
|Age at diagnosis||6 months||4 months||14 years|
|Age at study||3 years||2 years||16 years*|
|Presenting symptoms||Respiratory distress, lactic acidosis||Lactic acidosis||Vomiting, seizure, hypoglycemia|
|Mutation||c.1436A>T (p.Asp479Val)||c.1436A>T (p.Asp479Val)||c.685 G>T (p.Gly229Cys)|
|Eye||Normal||Nystagmus, not fixing or following||Horizontal oculomotor apraxia|
|Hearing||Normal||Conductive and sensorineural hearing loss||Right side severe sensorineural hearing loss|
|Cardiac involvement||Left ventricular hypertrophy||Normal||?|
|Bilateral inguinal hernia||Yes||No||No|
|Elevated liver transaminase||+||-||++++|
|Plasma amino acid||Normal||↑Alanine||Normal$|
|Brain MRI||Small basal ganglia infarcts||?|
|Treatment||Carnitine, thiamine||Not compliant with medications#|
Table 1: Clinical features of the patients with DLD deficiency. *Patient died at 16 years of age as result of hyperammonemia and liver failure post viral infection. $Normal branched-chain amino acids; in one test, allo-isoleucine was 3 nmol/mL (reference values, 0-2). #Lactic acidosis initially improved with thiamine; patient developed fever after taking lipoic acid.
The rate of cellular respiration (μM O2 min-1 per 107 cells) without treatment was 5.1 ± 0.9 (n=6). The corresponding rates in the presence of lipoic acid, thiamine, and carnitine were 8.1 ± 1.8 (n=6, p =0.002), 8.5 ± 1.6 (n=9, p <0.001), and 7.6 ± 2.0 (n=9, p =0.018), respectively. Thus, his fibroblast respiration was significantly lower (p =0.001) than that of control fibroblasts, and all treatment conditions resulted in improved respiration. Clinically, this patient responded to thiamine.
This infant also presented at birth with lactic acidosis (Table 1). PDHc activity in his fibroblasts was severely reduced. Sequencing DLD gene showed the same homozygous mutation c.1436A>T (p.Asp479Val). The rate of cellular respiration (μM O2 min-1 per 107 cells) in early passages without treatment was 5.7 ± 1.4 (n=6) . The rate of respiration in a late passage (passage #10) without treatment was 7.4 ± 1.4 μM O2 min-1 per 107 cells (n=6). The corresponding rates in the presence of lipoic acid, thiamine, and carnitine were 6.6 ± 1.6 (n=6, p =0.180), 5.1 ± 1.4 (n=9, p <0.026), and 5.0 ± 1.7 (n=9, p =0.008), respectively (Figure 3). Thus, his fibroblast respiration in the early passages was significantly low (p =0.001) compared to control fibroblasts. In contrast, his fibroblast respiration in the later passage was similar to control fibroblasts (p =0.051) and significantly decreased with the treatments. Clinically, this patient had severe manifestations of the disease and did not respond to any of the drugs.
This 16-year-old male presented with infection-triggered vomiting, hypoglycemia and elevated hepatic transaminases. He had mild learning disability; otherwise, he was asymptomatic (Table 1). The PDHc activity in his fibroblasts was 10.9 mU/UCS (reference range, 9.7 to 36). Sequencing the DLD gene showed the homozygous mutation c. 685 G>T (p.Gly229Cys).
Cellular respiration in his fibroblasts (passage #8) is shown in Figure 4. The rate of respiration (μM O2 min-1 per 107 cells) without treatment was 10.3 ± 3.3 μM O2 min-1 per 107 cells (n=6). The corresponding rates in the presence of lipoic acid, thiamine, and carnitine were 12.0 ± 2.3 (n=6, p =0.394), 13.6 ± 4.1 (n=9, p <0.181), and 15.5 ± 2.8 (n=9, p =0.012), respectively. Thus, although his fibroblast respiration was normal (p =0.836), it was significantly improved with the carnitine treatment. Clinically, this patient responded to carnitine.
The term “cellular bioenergetics” is defined as the biochemical processes involved in energy transformation, and the term “cellular respiration” is defined as the processes of delivering nutrients (catabolic metabolic fuels) and O2 to the mitochondria, oxidation of reduced metabolic fuels, passage of electrons to O2, and synthesis of ATP. Therefore, impairments in any of these processes will interfere with the rate of cellular respiration. Thus, DLD deficiency is expected to impair cellular bioenergetics and respiration [6-7].
Reference values for fibroblast respiration were recently reported . The rate was 9.8 ± 2.4 μM O2 min-1 per 107 cells (median=10.5, range=6.6 – 14.3, n=15). The two infants (Patients 1 and 2) with the homozygous mutation c.1436A>T had early-onset disease with severely reduced PDHc activities, but Patient 2 had a more severe phenotype than Patient 1. Their fibroblast respiration in early passages (#3) were also reduced (p =0.001). In Patient 1, the fibroblast respiration was significantly increased with all tested compounds (Figure 2). In Patient 2, the fibroblast respiration in the late passage (#10) was similar to control (p =0.051) and not increased with treatment (Figure 3). It is worth noting that the in vitro testing should be performed in the same cell culture passage, ideally in early passages (#3).
Patient 3 had a milder disease with normal PDHc activity, consistent with his homozygous mutation (c.685G>T) . His fibroblast respiration was normal in the fourth and sixth cell culture passages, but the lymphocyte respiration was low (data not shown). Thus, for disease screening, testing different tissues is recommended.
Lymphocyte respiration was measured in four patients with pyruvate dehydrogenase complex deficiency (Patients 1 and 2, and two other patients); the rate was 1.0 ± 0.62 μM O2 min-1 per 107 cells. The reference rate in 20 healthy children was 2.0 ± 0.9 μM O2 min-1 per 107 cells (median=2.0, and range = 0.9 – 3.7) [1,2]. Thus, lymphocyte respiration was reduced (p =0.067) in these patients compared to the controls.
It is worth noting that interacting metabolic reactions are necessary to maintain stability of the cellular bioenergetics. These processes require coordinating anabolic pathways in the nucleus and catabolic pathways in the mitochondria to create steady state energy potential in the cytosol . As previously suggested, inborn errors of metabolism impose imbalance in the catabolic and anabolic processes, which results in human diseases . In addition, mitochondrial dysfunction may result from caspase activation, which impairs cellular bioenergetics. However, this dependency on aerobic metabolic pathways may not apply to cells (e.g., cancer cells) that can maintain their survival on anaerobic metabolism (“aerobic glycolysis” or Warburg effect) . Thus, the biophysical changes in cellular bioenergetics (“capacitor operations”) in DLD deficiency and its clinical implications are expected to be complex and require investigations in future studies .
The results show cellular respiration is a suitable biomarker for DLD deficiency. The significance of using this tool to assess responses to therapies requires further studies. This approach may allow testing novel agents for disease treatment.
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