Sexual dimorphism in the cerebrospinal fluid total protein content

Introduction

Cerebrospinal fluid (CSF) is a clear, colorless body fluid filling the brain and the spinal cord. The CSF composition is very similar to a plasma ultrafiltrate with low protein concentration and few cells [1]. The diagnostic lumbar puncture, or spinal tap, is a mini-invasive procedure used to withdraw CSF from the spinal subarachnoid space [2]. With the only exception of brain biopsy, the CSF examination represents the only tool available to investigate the occurrence of (i) inflammatory, (ii) infectious, (iii) degenerative conditions, as well as (iv) the presence of CT-negative subarachnoidal haemorrhage and (v) of leptomeningeal metastases [3], [4].

CSF chemical and physical analyses are usually performed within 2 h from time of collection, and yield information on the fluid appearance, presence of discolorations, glucose content, circulating cell count and protein content [5].

Although the CSF/serum albumin ratio is considered the preferred marker to assess the blood-CSF-barrier (B-CSF-B) function [6], the determination of the CSF total protein (TP) content is – in routine clinical practice – an important screening test [7] for conditions such as bacterial meningitis, viral infections, autoimmune polyneuropathies, infectious polyneuropathies, subarachnoid hemorrhages and brain metastases [3].

An international agreement on the applicability of upper reference limits (URLs) for CSF-TP content is still lacking. A recently conducted web-based survey has disclosed that 45 mg/dL remains the most commonly used URL worldwide [8]. Higher CSF-TP content is found mainly in infants (<1 year) and premature babies [9], [10] due to an immature B-CSF-B, and in the elderly [3], [11], yet less than 5% of the participating centers reported the use of age-adjusted reference limits for adults and/or newborns [8].

Studies on the CSF/serum albumin ratio have recently demonstrated an increase in B-CSF-B permeability to proteins in men compared to women [12], [13]. Although this may lead to a difference in CSF protein content between the two sexes, to date only a few studies have explored this aspect [14] and above mentioned sex differences have not yet been considered in the daily laboratory practice.

With this work we aimed to address the effect of sex and age on the TP content in CSF obtained from diagnostic lumbar puncture, so as to provide some scientific ground for potential reconsideration of CSF-TP reference values.

Materials and methods

Results

Cerebrospinal fluid total protein concentrations ​​in the study population

The CSF-TP content median values ​​were significantly higher in men than in women in the overall study population (46 vs. 37 mg/dL) (Figure 1A). Stratifying the patients by age groups, the CSF-TP content was significantly higher in men than women in all age groups: 18–30 year (34 vs. 32 mg/dL); 31–40 years (44 vs. 34 mg/dL); 41–50 years (45 vs. 33 mg/dL); 51–60 years (51 vs. 41 mg/dL); 61–70 years (50 vs. 39 mg/dL); over 70 years (46.5 vs. 40 mg/dL) (Figure 1B).

Figure 1:

Cerebrospinal fluid (CSF) total protein (TP) levels in the study population analysed as a whole (A) and stratified by age (B).

Mann-Whitney U-test was used for all comparisons. CSF-TP content was higher in men than in women in the entire population (p<0.0001) (A) and in all age groups: 18–30 years (p=0.0474); 31–40 years (p>0.0001); 41–50 years (p<0.0001); 51–60 years (p=0.0016); 61–70 years (p=0.0002); >70 years (p=0.0015) (B). The line within the box indicates the median. The boundaries of the box represent the 25th–75th quartiles. The whiskers above and below the box correspond to the highest and lowest values.

In a multivariate model, natural log-transformed (ln) CSF-TP mean content (mg/dL) was significantly higher in men than in women even after adjusting for age at the time of lumbar puncture (3.80 vs. 3.59) and different hospital units of sample provenience (3.80 vs. 3.59) (Table 2).

Table 2:

Sex-specific mean (95% confidence intervals, CI) of the natural log-transformed (ln) cerebrospinal fluid (CSF) total protein (TP) content (mg/dL).

Men	Women	p-Valuea	Men	Women	p-Valueb	Men	Women	p-Valuec
3.807	3.583	<0.000001	3.801	3.589	<0.000001	3.804	3.589	<0.000001
(3.772–3.841)	(3.549–3.618)		(3.767–3.836)	(3.555–3.622)		(3.769–3.839)	(3.555–3.622)

1. Men = 604 (48.2%); Women = 648 (51.8%). Natural log-transformed (ln) distribution for CSF-TP content (mg/dL) and age at lumbar puncture (years). ^aStudent’s t-test for independent variables, crude means. ^bModel 1 (ANCOVA): marginal means adjusted for ln-transformed age at lumbar puncture. ^cModel 2 (ANCOVA): marginal means adjusted for ln-transformed age at lumbar puncture and hospital units of samples provenience. Bold p-values = statistically significant.

Correlation between age, sex and cerebrospinal fluid total protein

Age positively correlated to CSF-TP content in both women (Spearman, r=0.2086; p<0.0001) and men (Spearman, r=0.1540; p<0.0001). The difference between the slopes of the two sex-specific regression lines was not significant (F=0.007339; p=0.9317) while the difference between the elevations was significant (F=76.32; p<0.0001) indicating a constant gap of 8.5 mg/dL between men and women over the age of 18 (Figure 2).

Figure 2:

Linear regression analysis of cerebrospinal fluid (CSF) total protein (TP) content and age in men and women.

^aCSF-TP and age were positively correlated both in men (Spearman, p<0.0001) and women (Spearman, p<0.0001). ^bThe difference between the slopes was not significant (F=0.007339; p=0.9317) while ^cthe difference between the elevations was significant (F=76.32; p<0.0001).

Discussion

Our study shows that CSF-TP content is higher in men than women at all ages in a large population of adult patients undergoing lumbar puncture for diagnostic purposes. Being CSF-TP determination a widely used screening test for diverse neurological conditions [7], our findings show how abnormal CSF-TP content is more frequently reported for male than female individuals, irrespective of age.

The CSF-TP analysis is a laboratory tool commonly used in clinical practice, and a useful diagnostic support already in the earliest stages of the patient’s clinical condition, by virtue of the short time needed for analysis and report from sample withdrawal [3].

An increased CSF-TP content with age has been reported in the literature [7] which could be explained by an increase B-CSF-B permeability [6], [15], [16] and a progressive reduction of the CSF turnover both associated to ageing [1], [17]. The assessment of CSF-TP content has little diagnostic value in newborns’ neurological diseases due to the large variation for this parameter as a consequence of birth trauma and of the immaturity of the brain barriers [7], [10].

Recent studies propose age-dependent reference values ​​for CSF-TP content [8], [11], [14]. In particular, McCudden and colleagues highlighted that the URL of 0.45 mg/dL, currently the most used reference value worldwide, could produce 25% false positive patients aged over 65 years in the absence of neurological diseases [14]. In the attempt to reduce this ageing effect, some authors have proposed to use increasing URL values (e. g. 0.50 mg/dL for patients below age 30 and 0.60 mg/dL for older patients), but at present these thresholds have not yet entered into clinical practice anywhere in the world [8], [11].

Not only does age affect the CSF composition but sex too seems to play an important role. Despite higher contents of glucose, albumin, gamma globulins and TPs were reported in men as compared to women [18], these differences were never considered in setting the URLs for these analytes.

Recently, a 6 mg/dL difference was reported for CSF-TP content between men and women [14], yet sex-adjusted URL are highly overlooked in clinical practice.

Our present work is in line with this evidence, but we highlight a larger difference between the two sexes at all age groups, of both the median value and the linear regression coefficients, and of about 8.5/9.0 mg/dL. This observed difference from the cited work [14] appears to be partially artefactual due to the samples selection procedures. McCudden and coworkers excluded samples from patients with diseases that could increase of the CSF-TP content which however accounted only for nearly 19% of the pre-selected samples. In our work, while clinical diagnoses were not available for any of the samples, the hospital unit of provenience was known for each sample and the analysis was adjusted for this covariate which had no impact on the effect of sex over the CSF-TP content.

Moreover and to the best of our knowledge, we show for the first time a nearly fixed sex difference of CSF-TP content over the age of 18, and with parallel increase with age.

Proteins cross through the B-CSF-B by receptor-mediated transcytosis/endocytosis or by fluid phase absorption [19], [20]. Although no sex-related differences have been reported in the literature for these mechanisms, we now know that the B-CSF-B permeability to proteins is greater in men than in women, regardless of age and disease [12], [13], a mechanism which is likely ascribed to female hormones [21], [22] modulating a different expression of enzymes involved in the remodeling and turnover of the B-CSF-B [23], [24].

Confirming the effect of sex on the CSF-TP content, our study reiterates the need to re-define the application of the URLs for this analyte, which should be normalized for age and sex, in order to overcome the overestimation of CSF-TP content in men or, conversely, the underestimation in women, regardless of the age. Interestingly, in our study population, the use of URLs normalized by age and gender according to McCudden and co-workers [14] reduces this sex gap only partially.

Our study has some limitations. Due to the invasive nature of the lumbar puncture, which is performed for diagnostic or therapeutic purposes, our analyses did not include a population of healthy controls. To overcome such limitation, several studies have attempted to derive upper reference values ​​from patient samples [11], [25]. As in our study, the effect of peripheral blood contamination was minimized by removing the CSF samples with discolorations or increased WBC. Moreover, the lack of clinical diagnoses did not allow us to exclude patients with clinical conditions associated with a high CSF-TP content and this could account for the slightly higher CSF-TP levels as compared to studies conducted on “normal” subjects [14].

In conclusion, our study confirms a sexual dimorphism in the CSF-TP with a greater CSF-TP content more frequently assessed in men than in women, and that its values exceed the most commonly URLs references (45 mg/dL and 50 mg/dL).

In the era of precision medicine, better normalized CSF-TP reference values will have relevant implications on practice, adding further value to the CSF analysis, a preciously informative and quickly accessible ‘window’ to the CNS.

We encourage the use of sex- and age-normalized URLs in the daily CSF-TP analysis, as well as the search of the best sex- and age-specific threshold values by implementing a network of expertise and a larger study population, possibly including healthy CSF donors.