Lymphocyte respiration in children with Trisomy 21

Background This study measured lymphocyte mitochondrial O2 consumption (cellular respiration) in children with trisomy 21. Methods Peripheral blood mononuclear cells were isolated from whole blood of trisomy 21 and control children and these cells were immediately used to measure cellular respiration rate. [O2] was determined as a function of time from the phosphorescence decay rates (1/τ) of Pd (II)-meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. In sealed vials containing lymphocytes and glucose as a respiratory substrate, [O2] declined linearly with time, confirming the zero-order kinetics of O2 conversion to H2O by cytochrome oxidase. The rate of respiration (k, in μM O2 min-1), thus, was the negative of the slope of [O2] vs. time. Cyanide inhibited O2 consumption, confirming that oxidation occurred in the mitochondrial respiratory chain. Results For control children (age = 8.8 ± 5.6 years, n = 26), the mean (± SD) value of kc (in μM O2 per min per 107 cells) was 1.36 ± 0.79 (coefficient of variation, Cv = 58%; median = 1.17; range = 0.60 to 3.12; -2SD = 0.61). For children with trisomy 21 (age = 7.2 ± 4.6 years, n = 26), the values of kc were 0.82 ± 0.62 (Cv = 76%; median = 0.60; range = 0.20 to 2.80), p<0.001. Similar results (p<0.000) were obtained after excluding the five trisomy 21 children with elevated serum TSH (values >6.1 mU/L). Fourteen of 26 (54%) children with trisomy 21 had kc values of 0.20 to 0.60 (i.e., <−2SD). The values of kc positively correlated with body-mass index (BMI, R >0.302), serum creatinine (R >0.507), blood urea nitrogen (BUN, R >0.535) and albumin (R >0.446). Conclusions Children with trisomy 21 in this study have reduced lymphocyte bioenergetics. The clinical importance of this finding requires further studies.


Background
Trisomy 21 is the most common chromosomal anomaly worldwide, affecting about 1 in 700 newborns [1]. These individuals typically have low resting metabolic rates [2] and are particularly susceptible to infections [3] and hypothyroidism [4]. Moreover, defects in the inner mitochondrial membrane potential [5] and mitochondrial respiratory chain enzymes are documented in these patients [6,7]. Mitochondrial disturbances, increased oxidative stress and apoptosis have been described in the neurons, predisposing to precocious Alzheimer's disease [8]. Alterations in metabolic enzymes (e.g., monoamine oxidase-B, cytochrome oxidase, isocitrate dehydrogenase and glutamate dehydrogenase) have been also linked to impaired energy metabolism in trisomy 21 children [9]. Calcium levels are lower than in control children, which may alter cellular signaling [10].
Increased congenital heart disease and other major anomalies are exceptionally frequent in children with trisomy 21. It is not known whether these defects are linked to the biological impairments described above.
The use of the phosphorescence oxygen analyzer to measure lymphocyte respiration was recently reported. Lymphocytes were shown to be suitable for screening of certain mitochondrial disorders [11]. These methodologies were used to measure lymphocyte respiration rates in children with trisomy 21 and compare them with rates in children without this disorder.

Subjects
Venous blood samples (5 to 8 mL) were collected in heparin tubes and processed in <2 hr for peripheral blood mononuclear cell (PBMC) isolation and O 2 measurement. Blood was also collected from age-and gender-matched healthy controls. All trisomy 21 participants attended the outpatient facilities at Tawam and Al Ain Hospitals (Al Ain city, Abu Dhabi) for routine visits. All control participants were healthy children who had no medical complaints.
The study was approved by the institutional review board for protection of human subjects. Informed consent was obtained for each participating subject.

PBMC isolation
Plasma was collected from blood samples by centrifugation and possessed for Comprehensive Metabolic Panel and lipid profile. The samples were then diluted with equal volume of phosphate-buffered saline (PBS) containing 10 mM glucose and gently layered on the top of 10 mL Histopaque-1077. The mixtures were centrifuged at 15°C, 400 xg for 30 min. Collected PBMC were diluted with the same solution and re-centrifuged as above. The pellets were suspended in PBS, 10 mM glucose, 3 μM Pd phosphor and 0.5% fat-free bovine serum albumin for O 2 measurements at 37°C. Cell count and viability were determined by light microscopy, using a hemocytometer under standard trypan blue staining conditions. Only trypan blue-negative cells (>95%) were counted.

Oxygen instrument
A phosphorescence oxygen analyzer that measures dissolved O 2 in solutions as function of time was used to determine the rate of PBMC respiration [12,13]. This method is based on the principle that O 2 quenches the phosphorescence of a palladium phosphor [14].
The Pd (II) derivative of meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin had an absorption maximum at 625 nm and a phosphorescence emission maximum at 800 nm. Samples were exposed to light flashes (10 per sec) from a pulsed light-emitting diode array with a peak output at 625 nm. Emitted phosphorescent light was detected by a Hamamatsu photomultiplier tube after first passing it through a wide-band interference filter  Table 1) and control subject (Panel b). The lines are best linear fits (R 2 >0.830). The additions of 10 mM NaCN and 50 μg/mL glucose oxidase are shown. centered at 800 nm. Amplified phosphorescence was digitized at 1-2 MHz using an analog/digital converter (PCI-DAS 4020/12 I/O Board) with 1 to 20 MHz outputs.
The phosphorescence decay rate (1/τ) was characterized by a single exponential; I = Ae -t/τ , where I = Pd phosphor phosphorescence intensity. The values of 1/τ were linear with dissolved O 2 : where 1/τ = the phosphorescence decay rate in the presence of O 2 , 1/τ o = the phosphorescence decay rate in the absence of O 2 , and k q = the second-order O 2 quenching rate constant in sec -1 μM -1 (14). For calibration, the reaction contained PBS, 3 μM Pd phosphor, 0.5% fat-free albumin, 50 μg/mL glucose oxidase and various concentrations of β-glucose [11].
Cellular respiration was measured at 37°C in 1.0-mL sealed vials. Mixing was carried out with the aid of parylene-coated stirring bars. The respiratory substrates were endogenous metabolic fuels supplemented with glucose.

Statistical analysis
The data are summarized by arithmetic mean and standard deviation. Mann-Whitney U test was used for nonparametric values. P<0.05 was considered significant.  Figure 1a In children with trisomy 21 and normal TSH (n = 21), the k c value did not correlate with the TSH level (R 2 >0.072, Figure 3a). By contrast, in children with trisomy 21 and abnormal lymphocyte respiration (k c < 0.61, n = 14), the k c value correlated with the TSH level (R 2 >0.225, R >0.474, Figure 3b).
There were 8 children with trisomy 21 and congenital heart disease. Their median k c value was 0.6 μM O 2 per min per 10 7 cells (range, 0.2 to 1.6), and did not significantly differ from the remaining children (p = 0.238).   Table 1).

Discussion
The rates of lymphocyte respiration in the children with trisomy 21 were slower than in the control group ( Figure 2). These differences could reflect a relatively lower rate of mitochondrial energy conversion in trisomy 21 children that may be linked to some pathological findings pertinent to this disorder, such as defects in the inner mitochondrial membrane [5,8].
The mechanism for slower rates of lymphocyte respiration in children with trisomy 21 could be multifactorial. For example, the thyroid hormone is a well known regulator of the rate of metabolism; and hypothyroidism is common in children with trisomy 21. As shown in Figure 3b, high TSH (low or ineffective thyroxin) may contribute to the slower lymphocyte respiration in some children. Thus, thyroxin replacement is expected to improve lymphocyte respiration in those with hypothyroidism.
Of note, normal TSH values for children 2 to 7 years of age are 0.10 to 5.9 mU/L (mean = 2.2 mU/L) and for children 9 to 16 years of age are 0.20 to 6.1 mU/L (mean = 2.3 mU/L). Using these cutoffs, lymphocyte respiration was found to be higher in euthyroid trisomy 21 children than those with hypothyroidism.
Body-mass index, protein metabolism (BUN, total protein and albumin), and serum creatinine positively correlated with rates of lymphocyte respiration, but only in trisomy 21 children (Table 2 and Figure 4a-b). As previously reported, protein metabolism (proteolysis, oxidation and synthesis) is linked to obesity [15,16], a finding that is common in children with trisomy 21.
Close correlations were documented between cerebral O 2 consumptions and mental function, including depression and dementia [17,18]. It is unknown if our finding of slower lymphocyte respiration in trisomy 21 children is applicable to other organs. Nevertheless, our findings are consistent with the recent reports on  mitochondrial disturbances in those with trisomy 21 [19][20][21][22][23]. Decreased basal 3'-5'-cyclic adenosine monophosphate, increased reactive oxygen species and impaired NADH:ubiquinone reductase (complex I of the respiratory chain) were noted in fibroblasts from those with trisomy 21 [20].

Limitations of the study
No study was found in the literature that addressed lymphocyte respiration in children with trisomy 21.
Additional studies are needed in a larger population.

Conclusions
Children with trisomy 21 in this study have lower lymphocyte bioenergetics, a finding that is consistent with the known mitochondrial disturbances in these children. The clinical significance implication of this finding requires further studies.