Normal Appearing White Matter Metabolite Pattern and Sex Differences in Multiple Sclerosis Patients Compared to Healthy Controls

. Normal appearing white matter metabolite pattern and sex differences in multiple sclerosis patients compared to healthy controls


INTRODUCTION
Over the last 25-30 years, magnetic resonance imaging has been accepted as an important and essential method for specifying the multiple sclerosis (MS) diagnosis as well as for monitoring the disease in vivo. Proton magnetic resonance spectroscopy (PrMRS) is a method of investigating brain metabolites and the changes in their concentrations. The PrMRS is the only method specific for injuries of defi-nite cell types. [1] In MS patients, three types of metabolite patterns have been found: of acute and chronic plaques of demyelination, and of normal brain parenchyma (white brain matter outside the zones of demyelination). Studies over the last years have focused on the changes occurring in the brain parenchyma in MS outside the plaques of demyelination. In normal appearing white matter (NAWM) axon damage, without demyelination markers, microglia and astrocyte activation, and inflammation abnormalities of low intensity are found. MS is an autoimmune disease of genetic predisposition which in combination with factors from the environment, activates a cascade of immune responses and damages the blood-brain barrier. Inflammatory demyelination of the white brain matter of the central nervous system (CNS) occurs with the participation of T and B lymphocytes and macrophages. [2] Conduction of nerve impulses to the CNS is damaged which is clinically manifested as neurologic deficit. [3] The disease affects predominantly young people aged between 30-34 years. Twice as many females are disease-prone compared to males. [3] AIM The objective of the study was to measure the metabolite markers choline (Cho), creatine (Cr), and N-acetyl aspartate (NAA) in zones of NAWM (brain regions in the white matter without demyelination lesions) using PrMRS. The second goal of the study was to search for sex-determined differences in the parameters studied.

Proton magnetic resonance spectroscopy
MRT was performed on MT Signa 1.5T HDxt. The protocol for PrMRS we applied is used at the Department of Diagnostic Imaging of St Ivan Rilski University Multiprofile Hospital for Active Treatment, Sofia. PrMRS is a method in which signals are recorded reflected by the protons of organic molecules -metabolites in CNS. Their concentrations are 1000 times lower than those of water in tissues. The normal brain spectrum is presented by three resonance peaks of the metabolites. The first peak is formed by tetramethyl amines (Cho), which are mainly choline phospholipids. They participate in the synthesis and decomposition of cellular membranes. A second peak is formed by the content of Cr and phosphocreatine. They participate in the energy metabolism. The third peak is of the NAA groups which comprise the NAA localized within the neurons. A fourth peak may be formed, especially in pathologic processes, by lactates and lipids. [1] For the objective of our study, PrMRS was performed measuring Cho, Cr, and NAA in the zones of subcortical white brain matter free of demyelination lesions. Regions of normal Т2 image in the conventional MRI were selected, i.e. zones of NAWM. In the group of healthy controls corresponding zones of testing were selected. The data from the spectroscope measurement were compared. The frequencies of the metabolites tested were measured in units called parts per million (ppm) and they were graphically presented by peaks of different magnitude for the different metabolites (Fig. 1).

Patients
The study included 50 patients with specified MS diagnosis, according to the revised criteria of McDonald. [1,3] Patients with a history of the disease shorter than 10 years were selected, because of the fact that in 70% of the patients with RRMS the disease progresses to secondary progressive MS within 6-10 years after the onset. [1] The group of the patients studied was compared to a group of 28 healthy individuals, selected according to the corresponding demographic criteria.

Inclusion criteria:
• patients with clinically proven MS • age between 20-40 years • relapsing-remitting type of the disease • time elapsed since specifying the diagnose up to 10 years • currently receiving disease-modifying therapy interferon beta-1а [1,4,5] • Expanded Disability Status Scale (EDSS) up to 3.5 • with no evidence of relapse of the disease over the last 2 to 6 months [6] • with no administration of corticosteroid at least 2 months prior the study [6] Exclusion criteria: • relapse of the disease over the last 6 months prior to the study [6] • administration of corticosteroid over the last 2 months prior to the study [6]  • other autoimmune disease • history of specified diagnosis of depression, severe depressive episodes and/or treatment with antidepressants for symptoms of depression found • active inflammatory process • treatment with other type of disease-modifying therapy • motor deficit, visual or auditory disorders • alcohol or other psychotropic medications and substances abuse.

Exclusion criteria:
• history of other neurologic disease in the past -epilepsy, insult, encephalitis, meningitis, disorders of the CNS development • other autoimmune disease • history of specified diagnosis of depression, severe depressive episodes and/or treatment with antidepressants for symptoms of depression • active inflammatory process • alcohol and other psychotropic medications and substances abuse. All participants signed an informed consent. Demographic data were collected. Neurological examination was performed. PrMRS was performed for both groups. Cho, Cr, and NAA levels were measured in ppm in fronto-parietal NAWM. Corresponding zones of interest were selected in healthy controls. The data obtained were compared.

RESULTS
We studied 50 patients with clinically definitive RRMS. The distribution by sex was 29 (58%) female and 21 male (42%) MS patients. The individuals tested were 21-40 years old, with a mean age of 33±7 years, and duration of the disease of 4.85±2.68 years. The disease duration from the moment of diagnosis ranged between 5 months and 10 years. The control group counted 28 healthy controls. There were 14 female and 14 male subjects (50% : 50%), aged 27-40 years. The mean age was 33±5 years. All MS patients were employed on a full-time working day. There were 80% fulltime employed healthy controls, 6% students and 6% unemployed individuals in the control group. The MS patients were on disease-modifying treatment (mean duration of the treatment of 2.40±2.21 years). They were in a stable condition -remission at least 2 months prior to the tests, (minimal period of since on disease-modifying treatment 3 months, maximum period 8 years and 8 months). EDSS score ranged from 1.0 to 3.5. The distribution by EDSS score was as follows: 1.0 -16 patients (pts); 1.5 -10 pts, 2.0 -11 pts; 2.5 -11 pts; 3.0 -0 pts, and 3.5 -2 pts. Pyramidal functions deterioration was found in 18.5% of the MS patients. Cerebellar system symptoms were found in 8% and sensory symptoms in 7.5% of the patients. There were no abnormal signs with disability for upper limbs. Only 5% had brainstem symptoms and 4.2% had bowel-bladder dysfunction. There were no patients with visual or cerebral function disability included.
All patients and healthy controls underwent conventional MRI and PrMRS. Concentrations of brain metabolites in zones free of demyelination plaques (NAWM) were measured, respectively Cho, Cr, and NAA. Metabolic ratios NAA/Cr, NAA/Cho, Cho/NAA, Cr/NAA, Cho/Cr were registered, respectively. The data of the MRI were analyzed and compared to the data of the control group of healthy individuals in the respective age group (Fig. 2).
The data obtained were indicative of distinct changes in the metabolite concentrations with higher peaks registered in the MS patients.
Higher levels of Cho, Cr, and NAA were found in the MS patients. Statistically significant difference was found only when comparing the mean levels of Cr in healthy (controls) (х=61057±14334.29) and in patients (х=70228±17326.47) with a 5% risk of error. Kolmogorov-Smirnov test was ap- plied to check the normality of data distribution. The metabolites studied were not normally distributed. We applied the non-parametric Man-Whitney test for comparision (Table 1).
Only the result of the comparison of the mean values of Cr had a p-value (p=0.025) lower than the risk of error (α=0.05). Therefore, it could be said with 95% probability of certainty that there was a statistically significant difference between the healthy controls and the MS patients regarding the mean levels of this metabolite.
To read the clinical significance of the changes of the brain metabolites found comparative analysis of the changes in the ratios between the metabolites measured was performed, as follows: Cho/Cr, Cho/NAA, Cr/NAA, NAA/ Cho, and NAA/Cr. The comparision of the mean values of the NAA/Cr ratio in healthy controls and in MS patients found statistically significant difference respectively in healthy controls (х=2.59±0.46) and MS patients (х=2.36 ±0.45) with a 5% risk of error. In healthy controls, higher levels of the ratio were found.
The comparison of the mean values of the Cr/NAA ratio in healthy controls and MS patients found a difference in the mean values in healthy individuals (х=0.40±0.08) and MS patients (х=0.44±0.08), which was statistically signifi- When testing the ratios for normal distribution (Kolmogorov-Smirnov test) it turned out that only one ratio was normally distributed (NAA/Cr) and a parametric method of verifying the hypothesis about the difference between the mean values in MS patients and healthy controls was applied (Т-test) for it. In all the rest non-parametric test (Mann-Whitney test) was applied to verify the differences ( Table 2).
The results of comparison of the NAA/Cr ratio between healthy controls and MS patients had a p-value (p=0.035)smaller than the risk of error (α=0.05). Therefore, it could be claimed with a 95% probability of certainty that there was statistically significant difference between healthy controls and MS patients regarding the mean ratio of NAA/Cr metabolites.
The comparison of the Cr/NAA ratio between healthy controls and MS patients had a p-value (p=0.041) -smaller than the risk of error (α=0.05). Therefore, it could be claimed with a 95% probability of certainty that there was statistically significant difference between healthy controls and MS patients regarding the mean ratio of Cr/NAA metabolites. There was a change in all the ratios studied in the group of MS patients.
Statistically significant difference was confirmed only for the NAA/Cr and Cr/NAA ratios.
Twice as many females are disease-prone than males. [3] A possible impact of sex on the changes of brain metabolites was studied. The statistical difference assumed between the ratios of the metabolites Cho/Cr, Cho/NAA, Cr/NAA, NAA/Cho, and NAA/Cr studied was analyzed according to sex.

Males
The Kolmogorov-Smirnov test for normality of data distribution revealed that only the Cho/Cr ratio was not normally distributed and non-parametric method of testing the hypothesis about the difference between the mean values in MS patients and healthy controls was applied (Mann-Whitney test). In all the rest, parametric test (Т-test) was applied to verify the differences (Table 3).
No statistically significant difference was found when comparing the two groups of individuals studied.

Females
The difference between the mean values in female healthy controls (х=2.74±0.39) and female MS patients (х=2.38±0.45) was statistically significant with a 5% risk of error. In female healthy controls, increased levels of the ratio were found. The difference between the mean values in female healthy controls (х=0.44±0.08) and female MS patients (х=0.37±0.06) was statistically significant with 5% risk of error. In female MS patients, increased levels of the ratio were found.
When testing the ratios of normal distribution (Kolmogorov-Smirnov test), it turned out that NAA/Cr and Cho/ Cr were normally distributed. A parametric method of testing the hypothesis about the difference between the mean values in female MS patients and female healthy controls (T-test) was applied for them. In all the rest, non-parametric test (Mann-Whitney test) was applied to verify the differences ( Table 4).
The result of the comparison of the NAA/Cr ratio between female healthy controls and female MS pa- tients had a p=0.014 p-value, smaller than the risk of error (α=0.05). Therefore, with a 95% probability of certainty, it could be claimed that there was a statistically significant difference between healthy controls and MS patients regarding the mean ratio of the NAA/Cr metabolites. The result of the comparison of the Cr/NAA ratio between female healthy controls and female MS patients had a p=0.005 p-value, smaller than the risk of error (α=0.05). Therefore, with a 95% probability of certainty it could be said that there was a statistically significant difference between healthy controls and MS patients regarding the mean ratio of the Cr/NAA metabolites.
The results of the comparison between female healthy controls and female MS patients regarding the rest of the metabolites Cho/Cr, Cho/NAA, and NAA/Cho had a p-value greater than the risk of error (α=0.05). Therefore, with a 95% probability of certainty, it could be said that there was no statistically significant difference between healthy controls and MS patients regarding their mean ratios.
After verifying the difference between the changes found in the ratios between the brain metabolites and the sex of the individuals, it was found that only in females there was a statistically significant difference in the ratios studied.
Therefore, the female sex factor exerted considerable influence on the changes observed. The role of estrogen in the biosynthetic processes was to be considered.

DISCUSSION
Cho is a marker of cellular and membrane turnover. Therefore, an increased level of choline phospholipids is found. The reference about the stages preceding the appearance of a new plaque of demyelination in NAWM reports the increase of the levels of Cho metabolites. The data correspond to the changes found in NAWM in MS patients with PrMRS presented in studies of other research groups. [13] The increased levels of Cho reflect increased activity of Cho metabolism. Cho provides structural integrity and signal function of the cellular membranes. As a phospholipid component (phosphatidylcholine and sphingomyelin), it builds up the cellular membrane. It participates in the formation of the myelin sheath of the nerve fibers. [14,15] The changes found are in the zones free of demyelination. Regarding the levels of Cr, a difference is found between the two groups again with higher levels of the metabolite tested in the group of the MS patients. Cr is a marker of the energy homeostasis. The data obtained show increased energy exchange, i.e. intensity of the biochemical processes in NAWM in the MS patients tested. Statistically significant differences have been read only for Cr and that for the MS patients. [13] This fact can be interpreted as an increase of the energy utilization in NAWM. The increased energy resources and activity are probably related to a necessity of conducting an electrical signal under the conditions of changed ion homeostasis [16] -role of the virtual hypoxia [17,18] in the demyelinated axons, and they are also a testimony of active metabolite biosynthetic processes. Cr is a marker of energy reserve, and NAA -of neuronal integrity. The data obtained show statistically significant changes in the energy homeostasis and neuronal and glial integrity. From the analysis of the changes, it could be assumed that there is an increased energy exchange in the NAWM zones of interest, considering the maintenance of neuronal integrity. Previous studies showed decreased NAA/Cr ratio within the plaques of demyelination in MS patients. Decreased NAA/Cr and elevated Cho/NAA ratios in NAWM in MS patients when compared to healthy controls were also reported. [13,19,20] The findings were registered at an early stage of the disease development. [21] Decrease in NAA/Cr ratio is found also in normal appearing gray matter regions without inflammatory infiltration. [19] Comparative data analysis in our study revealed decrease in NAA/Cr ratio in RRMS patients which corresponds to data from published studies on metabolite changes in MS patients. [13,20,21] Statistically significant difference between both groups was found. The changes found in the brain metabolites and their ratios in NAWM show the presence of morphological changes in the white brain matter. These changes are an early indicator of breaking out of energy and structural disorders which, in turn, provoke disorders of the neuron-neuron communication and a potential of disconnection occurring. [22] An original study of brain tissue abnormalities in NAWM in MS patients treated with disease-modifying therapy and with no evidence of clinical activity of the disease is presented. An original comparative study of the metabolite pattern of NAWM in MS patients and healthy controls is presented. Sex-determined differences in the metabolite pattern of NAWM in patients with RRMS are found.

CONCLUSIONS
The use of PrMRS in our study proved metabolic changes in the regions free of demyelination (NAWM), which suggested the presence of parallel pathologic process prior to the appearance of demyelination plaque. Increased levels of Cho, Cr, and NAA in NAWM in patients with MS were measured. Statistically significant difference when comparing the NAA/Cr and Cr/NAA ratios in female MS patients was found.