Exploring differences in circulating metabolites of females and males with Alzheimer’s disease

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that leads to cognitive and functional decline and primarily affects the elderly population. Metabolic alterations, particularly in the amino acid and fatty acid pathways, are increasingly being recognized in AD. However, the role of sex in these metabolic changes remains insufficiently understood, despite evidence suggesting that AD may manifest more strongly in females. This study investigated sex-specific metabolic patterns in AD by analyzing routine and non-routine hematological tests, including amino acids and fatty acid profiles. The results showed that certain metabolites such as citrulline and alanine were frequently altered in patients with AD. Notably, docosahexaenoic acid, dihomo-gamma-linolenic acid, and gamma-linolenic acid levels were exclusively elevated in female patients. Additionally, females exhibited significantly lower Aβ42 and higher gamma-linolenic acid levels than males, with the trend becoming more pronounced during the early stages of the disease. Despite these differences, most metabolic markers did not show significant sex-based variation. These findings suggest that while some sex-specific metabolic differences exist in AD, a larger cohort is needed to confirm these patterns and fully understand the influence of sex on AD-related metabolic changes.

Keywords

Alzheimer’s disease Aβ42 alanine citrulline lipids

Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that represents the most prevalent form of dementia and is characterized by progressive memory loss, cognitive decline, and alterations in multiple physiological processes. The hallmark features of AD include the accumulation of amyloid-β plaques (Aβ) and neurofibrillary tangles, which lead to synaptic dysfunction, neuronal loss, and decline in cognitive abilities.^1,2

One emerging area of interest in AD research is the exploration of metabolic changes that occur in response to the disease or are closely linked to cognitive decline in AD. These changes often affect key metabolic pathways, such as amino acid and fatty acid metabolism, which have been shown to be disrupted in both animal models and human studies of AD.³

Although substantial research has focused on the clinical presentation and pathophysiology of AD, the role of sex in shaping metabolic changes associated with the disease remains an understudied but critical area.⁴ Epidemiological data indicate that females are disproportionately affected by AD, exhibiting higher prevalence rates and, in some instances, more rapid cognitive decline than males.^2,5 However, it is unclear whether these differences are primarily driven by biological sex differences or whether they reflect confounding factors such as differences in life expectancy, comorbidities, medications, or social determinants of health. Therefore, understanding whether sex-based differences in metabolism exist and how these differences influence AD progression is essential for the development of sex-specific diagnostic tools and therapeutic strategies.

The primary objective of our study was to identify potential hematological signatures that are differentially represented in males and females with AD. In particular, we focused on changes in amino acid and fatty acid levels which have recently gained significant interest in experimental and clinical studies on AD.^3,6 By investigating these factors, we aimed to contribute to the expanding body of knowledge surrounding sex-related dementia and identify new targets for therapeutic intervention.

Methods

Patients’ recruitment

Participants were consecutively recruited from the Center for Cognitive Disorders and Dementia (CDCD) and Center for Research and Training in Medicine of Aging (CeRMI) in Campobasso, Italy. Patients with probable AD were diagnosed according to the National Institute on Ageing/Alzheimer’s Association (NIA-AA) criteria.⁷ Patients with mild cognitive impairment (MCI) meet the NIA-AA diagnostic criteria for MCI.⁸ Clinical features of patients are reported in Table 1. To rule out other potential causes of cognitive impairment, all participants underwent the hematological tests reported in the Table 2 and brain imaging. Depression was ruled out using the Geriatric Depression Scale-Short Form (GDS-SF), and participants with a score ≥6 were excluded.⁹

Mini-Mental State Examination (MMSE) was used to evaluate global cognition, Basic Activities of Daily Living (BADL), and Instrumental Activities of Daily Living (IADL) to evaluate functional abilities, and the Cumulative Illness Rating Scale (CIRS) to assess patients’ comorbidities. The MMSE was performed to delineate the different stages of AD, with a score of 21 to 26 suggesting mild dementia (MD), 14 to 20 suggesting moderate dementia (MdD), and less than 14 indicating severe dementia (SD).

This study was conducted in accordance with the ethical principles of the Declaration of Helsinki and approved by the National and International Guidelines for Human Research. The Institutional Review Board (IRB) of the University of Molise approved this study (IRB Prot. N. 16/2020). Written informed consent was obtained from all the participants or caregivers.

Blood collection and analysis

Venous blood was collected between 7:30 am and 8:00 am, after overnight fasting for approximately 12 h. Blood tests, such as electrolyte, urea, creatinine, vitamin B12, and folate levels, are routinely used in the diagnosis of dementia. Other proteins, such as C-Reactive Protein (CRP), homocysteine, Antinuclear Antibodies (ANA), and anti-human Immunodeficiency Virus (HIV) antibodies, are utilized to exclude conditions that might simulate AD or signal an increased risk of cognitive decline.¹⁰

ELISA kit #ELK8920 (ELK Biotechnology, Denver, CO, USA) was used to assess Aβ42 levels. Aβ40 levels were not included in the analysis because they were below the sensitivity threshold of the kit in a substantial number of participants.

Two patterns were analysed: sex-specific differences and deviations from standard reference ranges.

Statistical analyses

Descriptive statistics, including means and standard deviations, were calculated using GraphPad Prism version 10.4.1. Fisher’s t-test or chi-square test was used to compare categorical variables between males and females. Spearman’s correlation coefficient was used to investigate the correlation between the amino acid and fatty acid levels and other blood signatures, using Bonferroni’s correction for multiple comparisons. Tukey’s or Sidak’s method was applied to ANOVA or multiple unpaired t-tests, respectively, for multiple comparison correction. Details of the statistical methods applied are provided in the table legends.

Results

We examined 121 individuals with AD, including 60 females (ADF) and 61 males (ADM), aged 62–92 and 62–90 years, respectively. All participants were Caucasian with a minimum of 12 years of education. Comprehensive data on age, body mass index (BMI), and key AD risk factors, such as smoking, alcohol consumption, and previous illnesses, were collected (Table 1). Of note, aside from smoking, where a higher number of males reported smoking more than three cigarettes per day, no other risk factors differed significantly between the sexes, and no significant differences were observed in cognitive or functional abilities (Table 1). Furthermore, no significant differences in treatment regimens were observed between females and males, and the therapeutic approach was equally effective across both sexes without necessitating sex-specific modifications.

Table 1.

Clinical features of patients with AD.

	Females(n = 60)	Males(n = 61)	p value
Aβ42 (ng/ml)	0.07 ± 0.04	0.11 ± 0.06	0.0001
Age (years)	78.15 ± 7.48	78.13 ± 6.90	NS
BMI (kg/m²)	25.22 ± 5.63	25.71 ± 4.94	NS
Age of onset (years)	72.60 ± 13.46	73.23 ± 7.52	NS
Duration of disease (years)	3.17 ± 2.12	3.67 ± 2.80	NS
MMSE
>24 normal
24–20 mild dementia	16.56 ± 7.86	19.54 ± 7.00	NS
20–13 moderate dementia
<13 severe dementia
BADL (/6)	4.76 ± 1.57	4.52 ± 1.80	NS
IADL (/8)	4.21 ± 2.59	3.77 ± 2.69	NS
CIRS (IS)	1.39 ± 0.36	1.41 ± 0.39	NS
CIRS (IC)	1.43 ± 1.81	1.69 ± 2.19	NS
Comorbidities
Smoke	16/60	32/61	0.003
Dyslipidaemia	23/60	22/61	NS
Diabetes	14/60	11/61	NS
Hypertension	32/60	32/61	NS
TIA	3/60	7/61	NS
Ischemia	2/60	5/61	NS
MCI	13/60	23/61	NS
MD	12/60	13/61	NS
MdD	16/60	11/61	NS
SD	20/60	13/61	NS
Medications (n. of patients)
Antidepressant	18/60	13/61	NS
Antihypertensive	27/60	28/61	NS
Hypolipidemic	19/60	21/61	NS
Hypoglycaemics	12/60	6/61	NS
Anti inflammatory	0/60	2/61	NS
Antiacid	11/60	14/61	NS
Antiplatelet	18/60	23/61	NS
Supplements	27/60	28/61	NS

Data are expressed as mean ± standard deviation (SD) or number upon total (n/total). Fisher’s exact test or Multiple unpaired t-tests corrected for multiple comparisons using the Sidak method were used for comparative analysis. Aβ42: amyloid beta-42; BMI: body mass index; MMSE: Mini-Mental State Examination. BADL: basic activities of daily living; IADL: instrumental activities of daily living; CIRS-IC: Cumulative Illness Rating Scale for Comorbidities and CIRS-IS: Cumulative Illness Rating Scale for Severity; TIA: transient ischemic attack; MCI: mild cognitive impairment; MD: mild dementia; MdD: moderate dementia; SD: severe dementia; NS: non-significant.

Most of the haematological parameters assessed revealed no significant sex-specific differences after correction for multiple comparisons (Tables 2 and 3), with the exception of Aβ42 levels, which were significantly higher in ADM (0.11 ± 0.06 vs. 0.07 ± 0.04; Table 1), and gamma-linolenic acid levels, which were significantly higher in ADF (26.1 ± 11.6 vs. 17.6 ± 8.2; Table 3).

Table 2.

Blood metabolites expressed in males and females with AD.

	Female (mean ± SD)	Male (mean ± SD)	p value
Basal blood glucose60–105 mg/dl	104.10 ± 23.30	102.40 ± 9.55	NS
GPT0–37 UI/L	19.25 ± 8.07	18.90 ± 5.12	NS
GOT0–45 UI/L	20.02 ± 10.0	19.28 ± 5.00	NS
Reuma Test0–20 UI/ml	16.51 ± 4.25	16.43 ± 3.00	NS
Urea20–50 mg/dl	46.59 ± 13.61	47.28 ± 10.22	NS
Creatinine0.7–1.3 mg/dl	0.87 ± 0.18	1.01 ± 0.21	NS
Uric Acid3.5–8.5 mg/dl	4.95 ± 1.35	5.66 ± 1.32	NS
Alkaline PhosphataseMale 53–128 U/LFemale 42–141 U/L	84.6 ± 19.9	85.5 ± 21.2	NS
Creatine phosphokinase (CPK)38–171 UI/L	95.61 ± 51.46	102.00 ± 34.59	NS
Amylase30–110 U/L	78.5 ± 26.8	78.0 ± 17.5	NS
Lipase8–57 U/L	27.7 ± 8.2	28.3 ± 5.5	NS
Blood cell count
Total Proteins6.3–8.2 g/dL	6.34 ± 0.36	6.50 ± 0.35	NS
WBC3.60–11 10³/ml	6.26 ± 2.08	7.01 ± 2.15	NS
NE%37–70%	60.70 ± 7.83	62.92 ± 9.58	NS
LY%20–45	29.98 ± 6.99	26.93 ± 8.32	NS
MO%1.1–12	6.62 ± 1.79	7.31 ± 1.59	NS
EO %0.1–8.0	2.45 ± 2.09	2.63 ± 1.62	NS
BA%0–3	0.25 ± 0.10	0.27 ± 0.17	NS
NE1.4–7 10³/ml	3.94 ± 1.47	4.51 ± 1.94	NS
LY1.2–3.4 10³/ul	1.87 ± 0.53	1.79 ± 0.55	NS
MO0.1–1.2 10³/ml	0.42 ± 0.16	0.51 ± 0.22	NS
EO0.00–0.80 10³/ml	0.15 ± 0.19	0.17 ± 0.11	NS
BA0.00–0.30 10³/ml	0.02 ± 0.01	0.02 ± 0.009	NS
RBC4.20–5.40 10⁶/ml	4.65 ± 0.44	4.96 ± 0.59	NS
HGB14–18 g/dL	13.58 ± 1.23	14.62 ± 1.81	NS
HCT42–52	41.40 ± 3.72	44.35 ± 5.25	NS
MCV80–99 fL	89.07 ± 5.93	89.45 ± 5.25	NS
MHC27–33 pg	29.20 ± 2.12	29.51 ± 2.29	NS
MCHC31.0–37.0 g/dL	32.79 ± 0.74	32.96 ± 0.90	NS
RDW11.5–15.5%	14.25 ± 1.34	14.24 ± 1.17	NS
PLT120–450 10³/ml	247.20 ± 54.01	218.10 ± 42.26	NS
MPV7.4–10.4 fL	9.53 ± 1.04	9.45 ± 0.89	NS
Albumin4.02–4.76 g/dL	3.86 ± 0.26	3.95 ± 0.35	NS
α1 globulin0.21–0.35 g/dL	0.26 ± 0.03	0.27 ± 0.07	NS
α2 globulin0.51–0.85 g/dL	0.66 ± 0.10	0.64 ± 0.12	NS
β1 globulin0.34–0.52 g/dL	0.38 ± 0.05	0.38 ± 0.06	NS
β2 globulin0.23–0.47 g/dL	0.28 ± 0.06	0.30 ± 0.07	NS
γ globulin0.80–1.35 g/dL	0.88 ± 0.22	0.95 ± 0.24	NS
α/γ globulin1.10–2.40 g/dL	1.59 ± 0.23	1.58 ± 0.26	NS
C reactive protein0.0–5.0 mg/L	4.44 ± 1.75	5.08 ± 2.97	NS
Transferrin200–400 mg/L	294.80 ± 38.62	303.10 ± 70.16	NS
Ions
Magnesium1.6–2.6 mg/dL	1.84 ± 0.19	1.86 ± 0.18	NS
Calcium8.4–10.2 mg/dL	8.91 ± 0.26	8.87 ± 0.22	NS
Sodium136–154 mEq/L	141.40 ± 1.84	140.40 ± 1.82	NS
Potassium3.5–5.3 mEq/L	4.29 ± 0.42	4.36 ± 0.39	NS
Chloride98–110 mEq/L	101.50 ± 1.21	101.40 ± 1.27	NS
Phosphorus2.7–4.5 mg/dL	3.61 ± 0.62	3.29 ± 0.43	NS
Insulin<20 mcUI/ml	13.09 ± 4.06	13.42 ± 3.65	NS
C380–170 mg/dL	120.50 ± 24.69	119.80 ± 23.63	NS
C412–36 mg/dL	28.41 ± 4.17	29.15 ± 4.34	NS
Anti-cytoplasmic antibodies (c-ANCA) absent: <0.8; present: >1.2	0.33 ± 0.21	0.35 ± 0.22	NS
p-ANCA absent: <0.8; present: >1.2	0.43 ± 0.18	0.45 ± 0.17	NS
Anti-cardiolipin (ACLA) IgG<10	3.53 ± 1.99	3.71 ± 1.93	NS
Anti-cardiolipin (ACLA) IgM<7	3.33 ± 1.22	3.58 ± 1.24	NS
Extractable Nuclear Antigen (ENA) ScreeningNegative < 0.7Positive >1.0	0.30 ± 0.16	0.29 ± 0.19	NS
ENA screening antibodiesratio: Negative < 0.7Positive >1.0	<0.7	<0.7	NS
Ceruloplasmin20–60 mg/dL	25.21 ± 6.97	23.83 ± 3.90	NS
Vitamin E5.5–17 mg/L	9.82 ± 3.03	9.85 ± 2.87	NS
Lupus-like anticoagulantNegative <1.2Mild 1.2–1.5 Moderate 1.5–2 Severe > 2	0.98 ± 0.13	1.01 ± 0.09	NS
ProcalcitoninNormal value < 0.10 Low risk of sepsi <0.50 Increased risk of sepsi 0.50–2.0High risk of sepsi > 2.0	0.07 ± 0.1	0.09 ± 0.2	NS
LDH<480 U/L	312.20 ± 45.09	288.80 ± 40.25	NS
Lactic Acid5.7–22 mg/dL	19.12 ± 3.37	18.12 ± 3.54	NS
Pyruvic Acid60–100 mmol/L	82.23 ± 10.88	87.73 ± 12.54	NS
Vitamin A0.20–0.80 mg/L	0.52 ± 0.14	0.53 ± 0.10	NS
Vitamin B132–95 mcg/L	46.26 ± 12.45	44.95 ± 12.68	NS
Cobalamin (Vit B12) 179–1162 pg/ml	505.30 ± 237.60	524.00 ± 189.10	NS
Vitamin D15–130 ng/ml	24.45 ± 11.25	24.29 ± 7.13	NS
Folic Acid4.6–34.8 ng/ml	7.51 ± 3.82	7.83 ± 3.91	NS
Vitamin B63.6–18 mg/L	9.52 ± 3.65	11.70 ± 11.37	NS
Amino acids
Homocysteine6–15 mmol/L	13.78 ± 6.49	13.30 ± 2.99	NS
Aspartic Acid<15 mM	9.42 ± 6.85	8.23 ± 5.73	NS
Glutamine200–800 mM/L	568.50 ± 176.90	586.00 ± 162.80	NS
Glutamic Acid30–120 mM/L	116.30 ± 117.60	119.20 ± 144.80	NS
Ornithine50–200 mM/L	98.63 ± 41.14	99.38 ± 45.36	NS
Citrulline10–35 mM/L	38.88 ± 14.94	38.85 ± 10.45	NS
Asparagine25–60 mM/L	41.67 ± 1.0	44.41 ± 10.33	NS
Methionine10–50 mM/L	25.06 ± 9.65	28.02 ± 8.60	NS
Serine35–190 mM/L	113.60 ± 46.64	108.50 ± 26.27	NS
Glycine120–340 mM/L	268.60 ± 69.59	236.0 ± 65.73	NS
Threonine40–220 mM/L	139.20 ± 38.27	136.90 ± 36.97	NS
Histidine40–140 mM/L	79.13 ± 20.93	81.69 ± 25.36	NS
Alanine150–400 mM/L	413.80 ± 116.80	417.80 ± 119.40	NS
Taurine20–100 mM/L	63.02 ± 19.85	58.50 ± 16.75	NS
Arginine30–90 mM/L	77.05 ± 24.18	69.16 ± 20.66	NS
α-aminobutyric acid10–40 mM/L	20.91 ± 7.51	21.40 ± 7.27	NS
Tyrosine30–200 mM/L	71.35 ± 37.14	73.66 ± 21.11	NS
Valine150–320 mM/L	225.40 ± 43.25	241.40 ± 49.46	NS
Tryptophan5–40 mM/L	25.22 ± 25.49	25.11 ± 25.18	NS
Phenylalanine30–80 mM/L	61.63 ± 16.46	63.57 ± 15.51	NS
Isoleucine30–100 mM/L	79.00 ± 28.94	89.31 ± 29.36	NS
Leucine50–190 mM/L	125.80 ± 35.68	136.20 ± 34.59	NS
Lysine80–300 mM/L	148.60 ± 61.32	138.80 ± 61.86	NS

Multiple unpaired t-tests corrected for multiple comparisons using the Sidak method were used for comparative analysis. Data are exspressed as Mean ± SD. GOT, glutamic-oxaloacetic transaminase; GPT, glutamate pyruvate transaminase; WBC, white blood cells; NE, neutrophils; LY, lymphocytes; EO, eosinophils; MO, monocytes; BA, basophils; RBC, red blood cell; HGB, haemoglobin; HT, haematocrit test; MCV, mean corpuscular volume; MCHC, mean corpuscular haemoglobin concentration; MCH = Mean Corpuscular Haemoglobin; RDW, red cell distribution width; PLT, platelets; MPV, mean platelet volume; LDH, lactate dehydrogenase; NS, non-significant.

Table 3.

Lipids expressed in males and females with AD.

	Female(mean ± SD)	Male(mean ± SD)	p value
Saturated fatty acids of serum/plasma lipids
14:0 Myristic Acid24–64 mg/L	35.03 ± 17.12	27.83 ± 15.27	NS
16:0 Palmitic Acid609–893 mg/L	870.10 ± 270.40	784.70 ± 189.40	NS
18:0 Stearic Acid186–279 mg/L	280.20 ± 69.45	247.10 ± 59.50	NS
20:0 Arachidic Acid5.0–9.0 mg/L	7.34 ± 3.08	5.89 ± 1.86	NS
22:0 Behenic Acid14–26 mg/L	23.15 ± 11.97	20.14 ± 7.88	NS
Omega-7 and Omega-9 fatty acids of serum/plasma lipids
16:1w7 Palmitoleic Acid48–124 mg/L	82.66 ± 38.58	61.71 ± 34.75	NS
18:1w9 Oleic Acid593–916 mg/L	1042.00 ± 394.90	920.20 ± 284.80	NS
24:1w9 Nervonic Acid24–41 mg/L	38.59 ± 17.58	31.19 ± 10.50	NS
Omega-3 fatty acids of serum/plasma lipids
18:3w3 Alpha-linolenic Acid13–33 mg/L	14.11 ± 7.72	12.60 ± 5.53	NS
20:5w3 Eicosapentaenoic Acid (EPA) 16–61 mg/L	24.99 ± 12.03	23.22 ± 12.58	NS
22:6w3 Docosahexaenoic Acid (DHA) 44–111 mg/L	115.80 ± 51.09	110.80 ± 41.99	NS
Omega-6 fatty acids of serum/plasma lipids
18:2w6 Linoleic Acid781–1167 mg/L	1117.00 ± 423.10	938.80 ± 229.10	NS
18:3w6 Gamma-linolenic Acid (GLA)8–25 mg/L	26.09 ± 11.59	17.56 ± 8.18	0.0001
20:3w6 Dihomo-gamma-linolenic Acid (DGLA) 37–75 mg/L	87.07 ± 31.80	68.64 ± 20.81	NS
20:4w6 Arachidonic Acid (AA) 188–343 mg/L	459.50 ± 149.80	386.20 ± 134.30	NS
Quotients
Saturated/unsaturated fatty acids ratio (0.4–0.6)	0.41 ± 0.06	0.43 ± 0.06	NS
Omega-6/Omega-3 fatty acids ratio (5.0–14.0)	11.54 ± 3.47	10.53 ± 3.36	NS
Arachidonic Acid/Eicosapentaenoic Acid ratio (AA/EPA)(<4)	22.54 ± 14.63	19.94 ± 8.45	NS
Omega-3 Index(6.0–8.0)	3.54 ± 1.21	3.84 ± 1.33	NS

Multiple unpaired t-tests were used for the comparative analysis. Statistical significance was corrected for multiple comparisons using Sidak’s method.

Stratification by clinical diagnosis and disease progression did not reveal any additional sex-related metabolic differences (Supplementary Table 1). However, several metabolites, particularly those within the lipidomic profile, showed notable increases in ADF when compared to standard physiological levels across the different stages of AD. Although these changes did not reach statistical significance across disease progression (Supplementary Table 1), they may reflect a sex-specific vulnerability in lipid metabolism, which appears more pronounced in females. Consistently, oleic acid and arachidonic acid levels exceeded the normal range in 65% ADF and 33% ADM and 86% ADF and 53% ADM, respectively. In addition, docosahexaenoic acid (DHA), gamma-linolenic acid (GLA), and dihomo-gamma-linolenic acid (DGLA) exceeded the physiological range only in ADF, and GLA levels were significantly higher in females than males (Table 3).

In contrast, Spearman’s correlation analysis did not identify any individual metabolites that significantly differentiated between sexes in relation to disease severity, as assessed by MMSE scores. Nonetheless, MMSE scores showed strong correlations with both IADL and BADL scores, reinforcing their value in reflecting functional decline in AD (Supplementary Table 2). This finding in accordance with previous studies conducted by our group,¹¹ may suggest physical activity as an effective non-pharmacological intervention that can be combined with pharmacological treatments to help delay cognitive decline in patients with AD.

Among the amino acids analyzed, citrulline and alanine levels exceeded the standard reference range in 63% of ADF and 68% of ADM, and in 52% of ADF and 60% of ADM, respectively. To evaluate whether the measurements provided by the analytical center were consistent with those typically found in blood samples from healthy elderly individuals (HLT), we assessed amino acid levels in a small subset of HLT participants. As shown in Supplementary Table 3, the amino acid levels in the HLT group fell within the established physiological range, and no significant sex-related differences were observed. Interestingly, correlation analysis in ADF and ADM revealed that citrulline was positively correlated with asparagine (r = 0.4, p = 0.0008) and threonine (r = 0.4, p = 0.0005) levels in ADF (Supplementary Table 4), and folic acid levels were negatively correlated with homocysteine levels in both ADF (r = −0.5, P = 0.001) and ADM (r = −0.5, P < 0.0004). However, the correlation between folic acid and homocysteine in ADF was not significant after the Bonferroni correction. It is well known that low folic acid levels may lead to homocysteine accumulation, which promotes neurotoxicity and vascular damage.^10,12,13

Discussion

The prevalence of AD is nearly twice as high in females as in males,¹⁴ with several sex-related risk factors contributing to this disparity, including age and life expectancy, the prevalence of cerebrovascular and cardiovascular diseases, and hormonal and reproductive changes in females.^5,15,16 These sex differences underscore the need for targeted prevention and intervention strategies to address the disproportionately higher burden of AD in women.¹⁷

To investigate the influence of sex on AD and its potential impact on disease progression, we conducted an extensive blood analysis of 61 males and 60 females with varying degrees of AD severity, ranging from MCI to SD. The study design incorporated standard reference values provided by the analytical laboratory to ensure accurate interpretation of the results in relation to potential pathological implications.

Overall, the comprehensive analysis of blood parameters revealed no significant sex-specific differences (Tables 2 and 3), with the exception of Aβ42, which was significantly lower in ADF, and GLA, which was significantly higher in ADF. A trend toward reduced Aβ42 concentrations in females (0.06 ± 0.03) relative to males (0.11± 0.06) was already pronounced at the MCI stage, despite comparable cognitive performance as indicated by similar MMSE scores (25.38 ± 2.48 vs. 24.95 ±2.34). Given that reduced peripheral Aβ42 may reflect increased amyloid plaque deposition in the brain,^5,18 these findings suggest that females might experience more advanced neuropathological changes in the early clinical stages of AD, potentially leading to faster clinical progression. This aligns with prior studies showing that the faster amyloid accumulation and accelerated disease progression in ADF is possibly due to more efficient Aβ42 clearance in males.^19,20

Several factors may underlie this observation, including alterations in liver and kidney function^21,22 and blood–brain barrier (BBB) permeability.^23,24 Traditional Chinese medicine also associates dementia with dysfunction in multiple organs, including the heart, spleen, lungs, kidneys, and liver.²⁵ However, our data did not reveal sex-specific differences in plasma markers associated with organ dysfunction. This suggests that the observed increase in Aβ42 burden in females is likely independent of systemic organ failure and may instead result from other mechanisms not captured by the current analyses, such as alterations in Aβ42 production, clearance, or aggregation pathways, all of which are crucial to disease progression.²⁶ Likewise, medication use is unlikely to explain these findings, as both sexes received the same pharmacological treatments (Table 1).

The risk of developing AD is particularly elevated in women following menopause, likely due to the decline in estrogenic levels, which are believed to have neuroprotective properties, coupled with women’s longer average life expectancy.⁴ Oestrogens have been shown to enhance Aβ42 clearance and suppress its production,^18,27–31 and Aβ42 accumulation has been observed during the perimenopausal and early postmenopausal periods.^32,33 Similarly, early menopause or surgical oophorectomy has been associated with increased amyloid burden.^34,35 Although we did not assess estrogenic levels in this study due to lack of hormone data, future research incorporating endocrine parameters in both sexes is warranted to better understand the role of sex hormones in the metabolic changes observed, especially in regard to the lipidomic profile that is known to be highly sensitive to fluctuations in estrogenic levels.³⁶ Indeed, we observed that females exhibited a stronger trend toward alterations in lipidomic pathways than males, particularly when stratified by clinical diagnosis. These changes were especially evident in metabolites involved in essential fatty acid metabolism, including linoleic acid, gamma-linolenic acid, and oleic acid, potentially reflecting sex-specific vulnerabilities in lipid regulation associated with AD pathology.

Some findings have suggested higher levels of inflammation in female.³⁷ However, in our study, no significant differences were found in inflammatory biomarkers. This finding may be due to the limitations of routine blood tests, such as C-reactive Protein, which may lack the specificity required to capture subtle inflammatory changes associated with AD.

Previous studies have highlighted the role of amino acids in aging and age-related dementia.³⁸ This study revealed notable alterations in circulating levels of citrulline and alanine in patients with AD, independent of sex. Citrulline plays a multifaceted role in AD, ranging from its involvement in the pathological mechanism of Aβ citrullination³⁹ to its putative use in managing conditions such as hypertension, endothelial dysfunction, and metabolic disorders.^38,40–43 Citrulline has been suggested as supplement to increase systemic arginine and NO levels in cardiovascular diseases and AD.^38,40–43 Additionally, emerging research has indicated that citrulline may influence energy metabolism and mitochondrial function,⁴⁰ further broadening its therapeutic potential. In our study, the finding of high circulating citrulline levels may challenge the use of citrulline in AD treatment. The trend in the increase in citrulline may be associated with dysregulation of nitric oxide (NO) production, which may contribute to oxidative stress and neuroinflammation, as previously suggested.⁴⁴ Furthermore, high citrulline levels may impair BBB function, promote neurovascular damage, reduce brain perfusion,⁴⁰ and trigger Aβ citrullination and accumulation.³⁹ Interestingly, when citrulline levels rise alongside other urea cycle intermediates, this could indicate impaired clearance of ammonia, which is neurotoxic and linked to AD.⁴⁵

In addition, we found that citrulline levels were positively correlated with threonine levels only in ADF. Citrulline is an amino acid produced primarily by enterocytes in the small intestine and is considered a reliable biomarker of intestinal function and mucosal integrity.³³ Threonine, an essential amino acid, plays a crucial role in maintaining gut health by contributing to the production of mucin, a key component of the intestinal mucus layer.⁴⁶ The observed correlation between these two metabolites suggests a possible interplay between intestinal barrier function and overall gut health and may indicate a possible intestinal dysfunction in ADF.

Elevated alanine levels may reflect abnormalities in glucose metabolism and mitochondrial function, leading to reduced ATP production and energy deficits in neurones.⁴⁷ The alanine aminotransferase (ALT) levels have been linked to increased Aβ deposition, reduced brain glucose metabolism, greater brain atrophy, and poorer cognitive performance.⁴⁸ In addition, elevated levels of alanine in AD brains, particularly in the gray matter, suggest the sequestration of amino acids in the brain and their potential role in AD progression.^49,50 Several studies have suggested that amino acid supplementation may increase amino acid levels in the brains of patients with AD and improve their cognitive performance.^45,51,52 However, evidence that plasma amino acids can pass the BBB remains controversial, and whether plasma alterations in alanine and citrulline reflect metabolic dysfunction rather than brain deficits^45,51,52 requires further investigation.

Interestingly, amino acid levels in the HLT group were within the physiological range, suggesting that the observed alterations in patients with AD may be linked to disease-specific mechanisms. However, the reduced sample size may have limited the statistical power to detect subtle differences, and this limitation should be considered when interpreting the results.

Our findings indicate that DHA, GLA, and DGLA exceed the physiological range in ADF. Additionally, GLA was higher in females than in males, suggesting a complex relationship between omega-3 and omega-6 fatty acids and AD, particularly in females. This relationship appears to be even more evident in the stratification of patients based on clinical diagnosis. Accordingly, we found that various studies have examined the effects of polyunsaturated fatty acids (PUFA) on cognitive function, particularly in relation to AD and MCI, with some suggesting potential benefits of PUFA in specific cognitive domains or early stages of cognitive decline, while others demonstrated limited or no significant effects. PUFA supplementation was reported to improve language ability and object location memory in healthy older adults,⁵³ but did not affect cognitive decline or depression in patients with MCI or AD.⁵⁴ Soininen et al., reported positive effects of long-term multinutrient intervention,⁵⁵ including PUFA, on cognition, function and brain atrophy in prodromal AD. Wu et al. meta-analysis demonstrated that higher fish consumption reduced AD risk⁵⁶ but found no statistical evidence linking PUFA to reduced dementia or AD risk. Zhu et al. suggested that higher PUFA intake may reduce MCI risk but found no significant association with AD or dementia risk.⁵⁷ The sex-specific differences in GLA levels in the present study could point to potential sex-specific mechanisms in AD pathology or progression and may inform future studies on targeted nutritional interventions or therapeutic approaches that consider the balance of omega-3 and omega-6 fatty acids, particularly in patients with ADF. However, given the complex and sometimes contradictory effects of fatty acids, such as linolenic acid on health, caution should be exercised when translating these findings into clinical recommendations without further investigation.

A significant limitation of this study is the small sample size, as a larger cohort of patients could have provided more robust subgroup analyses, potentially revealing important differences in metabolic profiles based on factors, such as disease severity, duration, or specific clinical manifestations. Therefore, while the current findings provide valuable preliminary insights, they should be interpreted with caution and considered as a foundation for future more extensive investigations in this field.

In conclusion, many routine and non-routine haematologic tests used in AD diagnostics and research did not show significant differences between ADF and ADM, except for Aβ42 being lower in females and gamma-linolenic acid being higher in females. These findings contribute to expanding our knowledge of sex differences in AD and underscore the complex interplay among sex, metabolism, and disease progression, thus providing a foundation for developing targeted diagnostic tools and therapeutic strategies for AD.

Supplemental Material

sj-pdf-1-jcb-10.1177_0271678X251340513 - Supplemental material for Exploring differences in circulating metabolites of females and males with Alzheimer’s disease

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X251340513 for Exploring differences in circulating metabolites of females and males with Alzheimer’s disease by Sara Serafini, Antonella Angiolillo, Gabriella Ferretti, Giulia Viviani, Carmela Matrone and Alfonso Di Costanzo in Journal of Cerebral Blood Flow & Metabolism

Footnotes

Data availability statement

Data supporting the findings of this study are available upon request from the corresponding author.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: PRIN #2022JJ3LZY to CM and PRIN #2022N8TFX to ADC, CM, and PRIN#20222ZB4HK to ADC.

Acknowledgements

We thank Clara Milone (University of Naples Federico II, Naples, Italy) for her help with data collection from patients with AD.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Author contributions

Serafini Sara: Methodology investigation, data curation, and analysis; Angiolillo A: Methodology, Data curation, and Resources; Viviani G: Data curation; Ferretti G: Methodology, investigation; Matrone C: Conceptualisation, resources, and writing the paper; Di Costanzo A: Conceptualisation, data curation, resources, and writing the paper. Serafini S, Angiolillo A, Ferretti G, Viviani G, Di Costanzo A and Matrone C: Reviewing and Editing.

Supplementary material

Supplemental material for this article is available online.

ORCID iD

Carmela Matrone

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