Delta-Aminolevulinic acid dehydratase enzyme activity and susceptibility to lead toxicity in Uganda’s urban children

Ambrose mukisa; Denis Kasozi; Claire Aguttu; Joseph Kyambadde

doi:10.12688/f1000research.108885.2

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Research Article

Revised

Delta-Aminolevulinic acid dehydratase enzyme activity and susceptibility to lead toxicity in Uganda’s urban children

[version 2; peer review: 1 approved, 1 approved with reservations, 1 not approved]

Ambrose mukisa¹, Denis Kasozi¹, Claire Aguttu¹, Joseph Kyambadde ¹

PUBLISHED 06 Jun 2024

Author details Author details

¹ Biochemistry and Sports Science Department, Makerere University, Kampala, Uganda

Ambrose mukisa
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation

Denis Kasozi
Roles: Data Curation, Formal Analysis, Software, Supervision, Visualization, Writing – Review & Editing

Claire Aguttu
Roles: Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – Original Draft Preparation

Joseph Kyambadde
Roles: Conceptualization, Funding Acquisition, Project Administration, Resources, Supervision, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background

With rapid industrialization, urbanization, and population explosion in sub-Saharan Africa including Uganda, the population has experienced increased exposure to environmental lead subsequently causing elevated blood lead levels. Mean blood levels of 332µg/dL,120µg/dʟ, 25µg/dL,11µg/dL, and 10µg/dL in children under 18 years of age in Nigeria, DR Congo, South Africa, Sudan, and Uganda respectively. Susceptibility to lead toxicity correlates with one’s nutrition status, age, and genetics. This study expounded susceptibility to lead toxicity by relating blood lead levels, delta-aminolevulinic acid dehydratase (ALAD) enzyme activity, and genetic variations of proteins that code for ALAD in urban children of Uganda aged between 6 and 60 months.

Methods

A total of 198 blood samples were analyzed for blood lead levels (BLL), on an atomic absorption spectrophotometer whereas hemoglobin (Hb) levels, and ALAD enzyme activity, were analyzed on a spectrophotometer before DNA extraction, polymerase chain reaction, and restriction fragment length digestion for ALAD polymorphism.

Results

Geometric means of BLL (10.55µg/dL, SD = 7.4), Hb (7.85g/dL, SD = 1.3) and ALAD enzyme activity (37.15 units/L BLL, S.D = 9.7), corresponded to samples that coded for ALAD1 allele (99.05%) compared to the 0.05% that coded for ALAD2 with BLL (14.5µg/ dL, SD = 4.7), Hb (6.1 g/ dL), ALAD enzyme activity (33.8 units/L, SD=1.45). There was a significant relationship with a negative linear correlation between BLL, Hb (status, and ALAD enzyme activity in the three isozymes (ALAD1-1, ALAD1-2, and ALAD2-2) in the strength of ALAD1-1 (r = 0.42, p-value = 0.02) ˂ ALAD1-2 (r = 0.62, effective size = 0.43, p-value = ˂ 0.001) ˂ ALAD2-2 (r = 0.67, effective size = 0.86, p-value = ˂ 0.001).

Conclusions

Most of the study participants coded for the ALAD1 allele hence hoarded blood lead, which could result in delayed exposure and adverse effects later in their lives.

Keywords

Blood Lead levels, Lead toxicity susceptibility, d-aminolevulinic acid dehydratase enzyme activity, d-aminolevulinic acid dehydratase gene polymorphism.

Corresponding author: Joseph Kyambadde

Competing interests: A preprint version of this article is available at https://www.preprints.org/manuscript/202112.0371/v1

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2024 mukisa A 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 work is properly cited.

How to cite: mukisa A, Kasozi D, Aguttu C and Kyambadde J. Delta-Aminolevulinic acid dehydratase enzyme activity and susceptibility to lead toxicity in Uganda’s urban children [version 2; peer review: 1 approved, 1 approved with reservations, 1 not approved]. F1000Research 2024, 11:538 (https://doi.org/10.12688/f1000research.108885.2) First published: 18 May 2022, 11:538 (https://doi.org/10.12688/f1000research.108885.1) Latest published: 24 Apr 2025, 11:538 (https://doi.org/10.12688/f1000research.108885.3)

Revised Amendments from Version 1

The revised version of this manuscript presents corrections as suggested by the reviewers. The latest version presents improved formatting, spacing, grammatical, and typo errors. In the abstract section, worldwide and local Lead toxic levels, the measurements for BLL, Hb, and ALAD that were missing in the original version are incorporated. The means and the standard deviation of the BLLs and Hb missed in version 1 were added. A Statement on Cohen’s d effective size was also included in the abstract. The background statements were rephrased following the reviewers' suggestions. The social demographic and the inclusion and exclusion statement of participating patients are included in the corrected version. Table 1 of version 1 missed the population aspect and this is addressed. The introduction of the latest version has been improved with the latest citations incorporated. All these corrections were included per the reviewers’ opinions.

To read any peer review reports and author responses for this article, follow the "read" links in the Open Peer Review table.

Introduction

Uganda, like many other African countries, is faced with rapid economic development, industrialization, population explosion, and urbanization.¹ These transitions are coming with both environmental and health challenges. Population explosion is putting pressure on the environment through increased anthropogenic activities, and elevated volumes of electronic wastes, and this has resulted in increased volumes of toxic pollutants like lead in both air and water bodies.¹ Because lead is an accumulative toxin, its increased concentration in the environment continues to cause health challenges, especially for children.²^–⁵ Lead is an environmental contaminant at high- and low-exposure levels.⁶^,⁷ However, levels that are deemed low enough to be safe are still detrimental to the developing central nervous system in children as lead is a neurotoxin with no safe level of exposure.⁷^–¹⁰ Elevated environmental lead levels usually correlate with the blood lead levels in exposed individuals.¹¹ Childhood lead exposure is associated with various health challenges that include lung, stomach, and bladder cancers, anemia, neurocognitive disorders, intelligence quotient (IQ) lowering, and stunted growth.⁵^,¹² Although environmental lead pollution is preventable, little attention is accorded to this preventable problem in many African countries including Uganda. Recent studies conducted in different parts of Kampala slums report elevated blood lead levels, especially among children.⁴^,¹³ One’s susceptibility to lead toxicity is modulated by age, genetics, nutrition, and malaria infection status.⁴^,¹⁴^–¹⁶ The rate of lead ion absorption, especially in the intestines, is further shown to increase with a decrease in hemoglobin levels. Following its absorption, approximately 98–99% of the lead in the bloodstream is bound to erythrocytes, where it exerts a destabilizing influence on cellular membranes.¹⁷

2 delta - ALA ⇄ porphobilinogen + 2 H_{2} O

Within red blood cells, lead reduces cell membrane flexibility and elevates the rate of erythrocyte breakdown, leading to anemia. Lead further specifically binds the delta-aminolevulinic acid dehydratase (ALAD) enzyme which is important in the heme biosynthetic pathway. This enzyme is involved in the condensation of glycine and succinyl CoA, and decarboxylation into delta-aminolevulinic acid (ALA) during the initial step of heme synthesis that takes place in the mitochondria before subsequent intermediate steps that take place in the cytoplasm and mitochondria again.

Porphobilinogen is formed by the combination of two molecules of δ-ALA with the help of the enzyme δ-aminolevulinic acid dehydratase (δ-ALAD) in the cytosol.

Subsequently, the enzyme ferrochelatase in the mitochondria facilitates the incorporation of a ferrous ion (Fe2+) into protoporphyrin IX to create heme. Delta-ALAD plays a vital role in lead poisoning, as its inhibition reduces heme production, leading to an increase in δ-ALA levels in the blood and urine of individuals exposed to lead. The synthesis of heme is not significantly affected until δ-ALAD activity is inhibited by 80–90%,¹⁸ which typically happens at a blood lead concentration of around 55 μg/dL.

The enzyme ALAD is rich with thiol groups and zinc ions, that have a high affinity for lead ions and this renders the enzyme more sensitive to attack by circulating lead ions.¹⁹^,²⁰ It is a tetramer homodimer with eight identical subunits and is located in the cytoplasm. In each of its subunits, it binds eight zinc atoms, where four zinc molecules act as catalysts, whereas the remainder serves as tertiary structural stabilizers. In times of lead burden, lead ions displace zinc from the enzyme’s active site and inhibit its activity, resulting in the accumulation of ALA.²¹ Accumulated levels of ALA trigger the production of reactive oxygen species (ROS), which are associated with oxidative stress.

Lead-induced oxidative stress results from the production of reactive oxygen species (ROS) and depletion of antioxidant reserves, particularly glutathione (GSH).²²^,²³ Lead interacts with GSH and antioxidant enzymes, inhibiting their functions and disrupting redox balance. Lead also interferes with essential cations, affecting various biological processes. Its ability to cross the blood-brain barrier and disrupt protein kinases can lead to neurological deficits.²⁴ Lead may induce DNA damage, inhibit repair mechanisms, and alter gene expression even at low concentrations.²⁵ Ingestion of lead ions can impact enzyme activities, protein levels, and blood parameters.¹⁴ Preventive antioxidant measures are crucial in mitigating lead-induced oxidative damage, as complete lead removal from the body is challenging.²⁶^,²⁷ The oxidative stress induced by lead triggers harmful chain reactions, leading to lipid peroxidation, protein and DNA damage, and potential carcinogenic effects.²⁸ Lead’s impact on cell membranes and signaling processes further exacerbates its toxic effects.

Several studies from different regions indicate varying blood lead levels, biological markers, and even symptoms among people in the same locality. This observation is attributed to the polymorphic nature of the gene that codes for the ALAD enzyme. Polymorphism of the ALAD gene is reported to modulate one’s susceptibility to lead toxicity.²⁹^,³⁰ The ALAD enzyme is encoded by a single gene on chromosome 9q34 region.³¹ This gene codes for two alleles i.e., ALAD-1 and ALAD-2,³² which are codominant (Single Nucleotide Polymorphism database (dbSNP) ID: rs1800435 [ http://www.ncbi.nlm.nih.gov/SNP/index.html]. Their expression results in a polymorphic enzyme system consisting of three different isozymes: ALAD1-1, ALAD1-2, and ALAD2-2. Individuals dominantly expressing ALAD1-2 and ALAD2-2 have a higher susceptibility to lead toxicity than those expressing the ALAD1-1 isozyme. The prevalence of the ALAD-2 allele is race-specific and usually ranges from 0 to 20 percent.²⁹ Therefore, the ALAD polymorphism affects and modifies lead metabolism and delivery to target organs.³³ To date, no study regarding ALAD enzyme activity and polymorphism distribution in the Ugandan population has been conducted. The present study, therefore, aimed at expounding on the ALAD enzyme activity, and the distribution of ALAD genotypes to lead exposure susceptibility in Ugandan children. Thus, this is the first study to address lead exposure susceptibility, ALAD enzyme activity, and polymorphism in Ugandan children.

Methods

Ethical considerations

This study was approved by Gulu University Research Ethics Committee No. (GUREC-048) dated 31/05/2019. The intentions of the study were first clearly explained in both English and a local language to the participant’s parents/guardians before signing informed consent forms.

The study design

This was a cross-sectional study that involved randomly selected children who resided in the Katanga slum of Kampala Uganda (00°18′49″N 32°34′52″E, coordinates) for at least a year. The area has approximately 7000 inhabitants and 15.2% of these are under 5 years of age [http:/www.askyourgov.ug]. Children aged between six and sixty months who had resided in the area for at least twelve months were included in the study. Those who had lived in the area for less than twelve months and were above the age of 5 years were excluded from this study.

The sample size (n) for the study was derived from Cochran’s sample size expression;

n = \frac{Z^{2} \cdot P \cdot (1 - P)}{e^{2}}

where p; is the population size, e; is the margin of error, and z; is the z-value, extracted from a z-table.

Through their local leaders, the homes of study participants were visited, and explained the purpose of the study was before the signing of consent forms by their parents/guardians. Visibly malnourished children and those with a history of blood transfusion were excluded from this study. Duplicate samples of venous blood (5 ml; n = 198) were drawn from each study participant by qualified nurses and technicians. One tube contained heparin and this was used for hematocrit determination, while the other tube containing ethylenediamine tetraacetic acid (EDTA) was used for other assays. The samples were transported on ice to the Makerere University Biochemistry Department laboratory for analysis.

Assay for blood lead using atomic absorption spectrophotometer

Blood lead levels were determined on an atomic absorption spectrophotometer (Agilent MY17180002 200 series) equipped with a graphite tube atomizer (GTA 120), a hollow-cathode lead lamp with a working current of 5 mA, 283.3 nm spectral line, and 0.5 nm bandwidth as described elsewhere.³⁴ Five hundred microliter (500 μl) aliquots of blood samples were mixed with 1.2 ml of a solution that was prepared by mixing equal volumes of 0.5% Triton X-100 and 1% di-ammonium phosphate ((NH₄)₂HPO₄). A total volume of 1.8 ml of deionized water was added to each sample in the tube followed by the addition of 1.5 ml of 20% trichloroacetic acid (TCA) before vortex mixing. The samples were centrifuged at 5000 rpm for 20 min and 10 μl of the supernatant from each was collected and injected into the graphite tube. Lead standard concentrations ranged from 2 μg/dL to 50 μg/dL. Samples were analyzed in duplicate, and their mean values were determined with occasional blanking with deionized/distilled water. The equipment had a detection limit of 2 μg/dL.

Colorimetric determination of hemoglobin levels by blood cyanmethemoglobin reaction method

Hemoglobin levels were determined following a cyanmethemoglobin reaction method described elsewhere.³⁵ Blood samples were processed and analyzed as described in our previous study.¹¹ Briefly, 100 μl of each sample was reacted with cyanide reagent and incubated at room temperature for 15 minutes. Hemoglobin concentration was then determined using a Jenway 6051 colorimeter at 540 nm against a reagent blank.

Determination of hematocrit levels of the study blood samples

The hematocrit levels of the study blood samples were assayed as described elsewhere.³⁶ Whole blood samples in heparinized tubes were forced into narrow-diameter glass capillary tubes to two-thirds levels. The capillary tubes had a self-sealing compound from one end. The capillaries together with the blood were loaded onto a micro hematocrit centrifuge and ran at a relative centrifugal force of 14,000 ×g for five minutes. Following centrifugation, hematocrit levels of each sample were measured within 10 min while the tubes were kept in a horizontal position to avoid merging of the layers. Hematocrit levels were estimated by calculating the ratio of the column of packed erythrocytes to the total length of the sample in the capillary tube.

Determination of delta-aminolevulinic acid dehydratase (ALAD) enzyme activity

The blood δ−ALAD enzyme activity in all the samples collected was measured following a method described by Ref. 37. The ALAD enzyme activity of each sample in duplicate was determined by incubating 0.20 ml of the sample with 1.30 ml of Triton X-100 reagent in disposable plastic tubes and thereafter adding 1 ml of buffered ALA substrate (0.01M). The buffered ALA substrate was prepared by dissolving 0.1676 g of ALA-HCL in 100 ml of phosphate-citrate buffer pH 6.65. The buffer was previously prepared by dissolving 6.703 g/dL Na ₂HPO ₄ (0.25 M) and citric acid 5.25 g/dL (0.25 M). Aliquots equivalent to 1ml of Trichloroacetic acid (TCA) reagent were added to each sample and the blank (plain distilled water).

To both test and blank aliquots, 1.0 ml of the modified Ehrlich’s reagent was added. This was previously prepared by dissolving 10 g of p-dimethylaminobenzaldehyde (DMBA) in 420 ml of acetic acid and diluted to 1 L with distilled water. Before storing the reagent at 40°C a working solution was prepared by mixing 50 ml of DMBA-acetic acid with 8 ml of 70% perchloric acid. Following the addition of the modified Ehrlich’s working reagent, the mixtures were allowed to stand for 13 min for color development before measurement at 555 nm on a spectrophotometer.

The corrected absorbance A = (Test absorbance – the blank absorbance) was used to calculate the activity of the enzyme.

\frac{Corrected Absorbance A \times 12500}{Hematocrit} = units of ALAD enzyme activity,

Where 12500 is the blood dilution factor.

Delta-aminolevulinic acid dehydratase (ALAD) genotyping

Blood samples were analyzed for polymorphism as described elsewhere,³⁸^,³⁹ Genomic DNA from each blood sample was extracted using a Qiagen genomic DNA purification kit (DNeasy, Catalogue no. 69506) following the manufacturer’s instruction. The resultant DNA products were purified before polymerase chain reaction (PCR) amplification. The PCR reaction mixture equivalent to 50 μL contained 1× buffer (10 mM Tris-HCl, pH 8.8; 50 mM KCl), 2 mM MgCl₂, 0.2 mM dNTPs, 20 pmol each primer, and 3U Taq DNA polymerase.

Forward primer, 5′-AGACAGACATTAGCTCAGTA-3′,

and reverse primer, 5′-GGCAAAGACCACGTCCATTC-3′

The running conditions on a Gene Amp PCR system 9700 were; pre-denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, synthesis at 72°C for 1min and final extension at 72°C for 5min. The amplified products (916-bp region of genomic DNA) in volumes of 10 μL were digested overnight with MspI restriction enzyme (2.5 units) in a 20 μL reaction mixture containing 50 mM sodium chloride, 10 mM Tris-HCl, 10 mM magnesium chloride, 1mM dithiothreitol (pH 7.9) at 37°C. The fragments were separated by electrophoresis on a 2% agarose gel stained with ethidium bromide and visualized under a UV illumination system. ALAD1-2 samples had both a 583- and a 512-bp fragment, whereas ALAD1-1 individuals had a single 583-bp fragment.

Data analysis

Results were expressed as means and correlations, and the statistical significance was evaluated by one-way analysis of variance (ANOVA) using Minitab 19 statistical software, an equivalent open-access alternative is Scilab-6.1.1 statistical software. In addition to maximizing data collection, missing data cases were completely omitted (list wise) from the data set before statistical analysis.

Results

Following genotyping of the samples for ALAD alleles, the outcome is shown in Table 1 with corresponding BLL, Hb levels, hematocrit, and ALAD enzyme activities. The results indicate that the ALAD1-1 isozyme was the most predominant with moderately high hemoglobin levels and seemingly normally functioning ALAD enzyme. The frequency of the ALAD2-2 isozyme is shown to be the least predominant as compared to the ALAD1-1 and ALAD1-2 isozymes. Comparing the hemoglobin levels across all the groups, it is apparent that members with ALAD2 allele have lower Hb levels compared to members coding for ALAD1.

Table 1.

The gene distribution of ALAD (delta-aminolevulinic acid dehydratase) isozymes and the corresponding blood lead levels, ALAD enzyme activity, hemoglobin, and hematocrit volume among the 198 study participants.

Isozyme	Frequency of ALAD isozymes among the study population (N)	Mean Blood lead levels (μg/dL)	Mean ALAD enzyme activity (Units/L)	Mean Hemoglobin levels (g/dL)	Mean Hematocrit volume (%)
ALAD1-1	0.889 (176)	8.8	39.6	8.9	27.6
ALAD 1-2	0.106 (21)	12.3	34.7	6.8	29.2
ALAD 2-2	0.005 (1)	14.1	33.8	6.1	32.9

The results further indicate that members with isozyme ALAD1-1 had their ALAD enzyme activity functioning moderately normal as compared to the rest. Correlational analysis revealed that ALAD enzyme activity and hemoglobin levels strongly correlated with blood lead levels across all the genotypes (Table 2).

Table 2.

Correlations between different ALAD (delta-aminolevulinic acid dehydratase) isozymes, blood lead levels, ALAD enzyme activity, hemoglobin levels, and hematocrit volumes.

Isozyme	Blood lead levels (μg/dL)	ALAD enzyme activity (Units/L)	Hemoglobin levels (g/dL)	Hematocrit volume (%)
ALAD 1-1	r = 0.42, p-value 0.02	r = 0.66, p-value ≤ 0.001	r = 0.51, p-value ≤ 0.001	r = 0.11, p-value ≤ 0.07
ALAD 1-2	r = 0.62, p-value ≤ 0.001	r = 0.71, p-value ≤ 0.001	r = 0.69, p-value ≤ 0.001	r = 0.16, p-value = 0.06
ALAD 2-2	r = 0.67, p-value ≤ 0.001	r = 0.71, p-value ≤ 0.001	r = 0.64, p-value ≤ 0.001	r = 0.12, p-value = 0.11

Discussion

The research explored the relationship between the activity of the ALAD enzyme and levels of blood lead, in Ugandan urban children aged 6-60 months. This age group was selected since it’s known to be more susceptible to even what would be safe levels of Lead than adults.⁷^,⁸^,¹⁰ The adverse effects of exposure to lead on human health are well known. It has also been reported that elevated environmental lead levels usually correlate with blood lead levels in exposed individuals.¹¹ This study found the majority of individuals sampled (99.5%) had the ALAD1 gene, while (0.05%) carried the ALAD2 gene which is in agreement with previous studies.³⁸^,⁴⁰^,⁴¹ There were significantly lower levels of BLL but higher Hb and ALAD enzyme activity in individuals who had the ALAD1 gene as compared to those with the ALAD2 gene (Table 1). Additionally, the study delved into how genetic differences in ALAD proteins impact the susceptibility to lead⁷ in children aged 6–60 months) living in Katanga, Uganda. The results showed a negative link between BLL, Hb levels, and ALAD enzyme activity among the three types of ALAD isozymes (ALAD1-1, ALAD1-2, and ALAD2-2). The correlation varied in strength, with ALAD2-2 displaying the strongest connection (r = 0.67, p < 0.001), followed by ALAD1-2 (r = 0.62, p < 0.001), and ALAD1-1 (r = 0.42, p = 0.02) (see Table 2). The observed differences in BLL among the study population are attributed to the differences in affinity for lead exhibited by both ALAD2 and ALAD1. ALAD2 binds lead more tightly, possibly increasing blood lead retention and associated toxic effects. The findings from the current study seem to contradict reports from some previous studies that indicate no significant difference in blood lead levels among different ALAD genotypes at low exposure levels. Because the study area (Katanga) is a city slum with social disadvantages, we speculate that other factors like nutritional status could have had a profound contribution to the observed study outcome. It was further observed that ALAD enzyme activity and hemoglobin concentrations showed significant differences across different ALAD genotypes i.e., ALAD1-1, 1-2, and 2-2 genotypes at low lead exposure levels. This concurs with studies that suggest that genetic variations in ALAD proteins influence the susceptibility to lead exposure²⁷^,⁴²^,⁴³ and that individuals with the ALAD2-2 isozyme may be more susceptible to the adverse effects of the exposure compared to those with ALAD1-2 and ALAD1-1.⁴⁴ The differences observed in BLL, Hb levels, and ALAD enzyme activity between individuals with ALAD1 and ALAD2 genes underscore the potential impact of genetic factors on lead toxicity and heme synthesis. The lower BLL and higher Hb levels in individuals with the ALAD1 gene may indicate more efficient lead detoxification and heme synthesis processes compared to those with the ALAD2 gene. Delta-aminolevulinic acid dehydratase (ALAD) is a key enzyme in heme production, converting delta-aminolevulinic acid (ALA) to porphobilinogen and being hindered by lead in the blood.⁴⁵^–⁴⁸ Genetic variations in ALAD, like the ALAD1 and ALAD2 genes, impact enzyme activity during lead exposure.⁴⁷ The diverse forms of ALAD, including ALAD1-1, ALAD1-2, and ALAD2-2, play a role in the susceptibility to lead toxicity.¹⁶^,²⁷^,⁴⁹ This could imply that individuals with the ALAD2 gene may be at a higher risk of lead-related health issues due to reduced detoxification capabilities and potentially compromised heme synthesis.

Hemoglobin levels are slightly higher in lead workers with the ALAD1-2 genotype, but the difference is not significant (reference). The presence of the ALAD2 allele is associated with a 4-fold increase in the ability to retain lead in the blood at levels above 30 μg/dL. ALAD2 carriers may tolerate higher and longer exposures to lead, potentially due to the allele’s higher affinity for lead. The effect of the ALAD genotype on blood lead levels is more pronounced in populations with high and prolonged lead exposure. There is no significant difference in ALAD enzyme activity between ALAD1 and ALAD2 carriers in terms of lead-induced inhibition.

These findings suggest that genetic variations in the ALAD gene could affect the activity of the ALAD enzyme, which in turn may influence susceptibility to lead toxicity and the production of heme in the body. The study emphasizes the importance of understanding how genetic and biochemical factors contribute to individual differences in lead toxicity and heme synthesis, which can have implications for health and disease risk later in life.⁴⁹ Further exploration in this field could offer insights into potential approaches to reduce lead exposure and its associated health impacts.

Conclusion

The study also highlights that the ALAD genotype is significantly linked to Hb levels, ALAD enzyme activity, and blood lead levels, with individuals carrying the ALAD2-2 gene showing higher lead levels compared to those with ALAD1-2 and ALAD1-1 genes. This underscores the significance of ALAD polymorphism in altering how the body processes lead and suggests the need for more extensive research involving a larger population in Uganda to gain a deeper understanding of how ALAD gene variations impact lead toxicity.

Study limitation

For better results on the effect of persistent exposure to low levels of lead, the study participants’ age range should have been from six months to eighteen years.

This study did not look at the nutritional status of the study participants which is important for lead toxicity susceptibility.

Data availability

Underlying data

This study’s participants were minors, whose data public sharing is ethically restricted. However, the data that support these study findings are available from the corresponding author [J.K., joseph.kyambadde@gmail.com], upon presenting a clear written statement on the purpose of the data request.

Extended data

Dryad: A representative gel of ALAD following a restriction fragment length digestion, https://doi.org/10.5061/dryad.vt4b8gttz.⁵⁰

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

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27. Qader A, Rehman K, Akash MSH: Genetic susceptibility of δ-ALAD associated with lead (Pb) intoxication: sources of exposure, preventive measures, and treatment interventions. Environ. Sci. Pollut. Res. Int. 2021; 28: 44818–44832. PubMed Abstract | Publisher Full Text
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Author details Author details

¹ Biochemistry and Sports Science Department, Makerere University, Kampala, Uganda

Ambrose mukisa
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation

Denis Kasozi
Roles: Data Curation, Formal Analysis, Software, Supervision, Visualization, Writing – Review & Editing

Claire Aguttu
Roles: Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing – Original Draft Preparation

Joseph Kyambadde
Roles: Conceptualization, Funding Acquisition, Project Administration, Resources, Supervision, Writing – Review & Editing

Competing interests

A preprint version of this article is available at https://www.preprints.org/manuscript/202112.0371/v1

Grant information

The author(s) declared that no grants were involved in supporting this work.

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mukisa A, Kasozi D, Aguttu C and Kyambadde J. Delta-Aminolevulinic acid dehydratase enzyme activity and susceptibility to lead toxicity in Uganda’s urban children [version 2; peer review: 1 approved, 1 approved with reservations, 1 not approved]. F1000Research 2024, 11:538 (https://doi.org/10.12688/f1000research.108885.2)

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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

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Reviewer Report 11 Nov 2024

Howard Hu, Department of Population and Public Health Sciences, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

Not Approved

https://doi.org/10.5256/f1000research.167147.r335186

The subjects of this report are novel and important, in that lead exposure remains one of the top environmental health problems in the world, whereas studies of lead exposure (with blood lead as a biomarker of exposure)

The subjects of this report are novel and important, in that lead exposure remains one of the top environmental health problems in the world, whereas studies of lead exposure (with blood lead as a biomarker of exposure) in Africa remain sparse. Moreover, the addition of analyses of the results across ALAD genotype, one of the most important known genetic modifiers of lead toxicokinetics, is quite novel. The overall manuscript and reporting are quite good, BUT there are several issues, mostly reflecting a lack of analyses and reporting format that align with epidemiology (and this is, essentially, an epidemiological study), some inconsistencies in the data, and some further improvements that are needed in terms of the writing. Details are provided below.
ABSTRACT
- 2^nd sentence: “Mean blood levels of 332 ug/dL, 120 ug/dL…”.
  - I would not call a mean blood lead level of 332 or 120 ug/dL as “elevated blood lead levels”---I think these should be identified as a examples of extreme clinical lead poisoning, whereas the mean blood lead levels of 25, 11, and 10 ug/dL are examples of elevated subclinical lead exposures.
  - The sentence itself is not a full sentence (no verb!).
- The methods section should include a few words describing the population sampled, e.g., “randomly selected children residing in the Katanga slum of Kampala, Uganda”.”
- The results (see discussion of results below)
INTRO
- In general, the discussion of lead’s molecular impacts could be shortened, if space is a limitation
- Last para: ALAD polymorphism is only one of several polymorphisms that have been associated with altered lead metabolism and/or susceptibility to toxic effects (examples of others: the VDR, HFE, Dopamine metabolism, etc.).
METHODS
- In general, excellent description of lab methods (which could be shortened if more space is needed)
- From epidemiological perspective: The authors should clearly state the number of individuals studied. It says “Duplicate samples of venous blood (5 mg; n=198) were drawn from each study participants…” but that that suggests the actual number of individuals was n=198, each having duplicate samples; or n=198 samples/2 = 99 individuals, each having duplicate samples. Re-word to say “Duplicate samples of venous blood (5 mg) were drawn from each study participants (n=198)…”
RESULTS
- From an epidemiological perspective, it is important to go over basic demographics (age, sex), data for which should be reported in the text as well as added to a revised Table 1. Given that there is only 1 individual with the ALAD 2-2 genotype, I would advise reporting this individual’s values in the text, but in the Table, merging this individual’s data with the ALAD 1-2 data and simply calling this group “ALAD 1-2 and 2-2” (it makes no sense statistically to analyze this one individual’s data as a separate category).
- I suggest that the variables are displayed in rows (not columns), the columns can take the form of “ALAD1-1”, “ALAD1-2 and 2-2”, and “Total”, and rows can be added at the top to report data on n (number of individuals in each group”, sex (Male v. Female); mean, SD, range of ages. .
- 99.5% of individuals have the ALAD1 allele (exclusively?); whereas 0.11% (NOT 0.05%) carried the ALAD2 allele. This also has to be corrected in the abstract
- The hematocrit results do not make sense. Hemoglobin and hematocrit level typically are highly correlated, whereas comparing the ALAD 1-1 with the ALAD 1-2 groups, the hemoglobin levels are lower whereas the hematocrit levels are higher, respectively. Such a discrepancy can sometimes occur, but only under unusual circumstance, such as if many of the individuals in the study had a hemoglobinopathy, such as Thalassemia, by which the hemoglobin levels are low, and the body tries to compensate by producing more red cells, which increases the hematocrit. The authors should either check their data to see if perhaps the values have been switched; or determine if hemoglobinopathy could be a major factor in this group of individuals, etc. Either way, they need to address this discrepancy in the discussion.
- Table 2: I would not analyze or describe these relationships as “correlations” with r-values. The differences in blood lead, ALAD enzyme activity, hemoglobin levels, and hematocrit across categories of ALAD genotype should be analyzed and reported as the results of ANOVA. In addition, all such results could easily be merged into Table 1.
- Regarding the data, I think there is a missed opportunity to use multivariate regressions to examine inter-relationships, such as treating hemoglobin levels or hematocrit levels as an outcome in relationship to (a) a model with age, sex, ALAD-1/1 or ½ versus wildtype (ALAD-1/1) as independent variables; and (b) a second model with age, sex, ALAD enzyme activity as independent variables.
DISCUSSION
- It would be more accurate to say that the 0.5% prevalence of ALAD2 is in agreement with other data specific to individuals in Africa, i.e., the study of Liberians by Benkmann et al (ref. 41).
- Consider adding discussion of the potential sources of the elevated lead exposure among these individuals living in this neighborhood of Kampala, particularly since lead exposure in low- and middle-income countries has recently become the topic of intense interest and activity by government agencies (such as UNICEF, US Agency for International Development, the World Bank) and NGO’s (such as Pure Earth).

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Environmental epidemiology, internal medicine, occupational/environmental medicine, toxicology.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Reviewer Report 14 Jun 2024

Lorenz S. Neuwirth, SUNY Neuroscience Research Institute, Old Westbury, New York, USA

Approved

https://doi.org/10.5256/f1000research.167147.r287537

Abstract:
In the abstract, the conclusion should contain more information containing a discussion of the results and then the conclusion. As it reads, it is incomplete.

Introduction:
It may just be the formatting of how ... Continue reading

Abstract:
In the abstract, the conclusion should contain more information containing a discussion of the results and then the conclusion. As it reads, it is incomplete.

Introduction:
It may just be the formatting of how the manuscript is set up, but the following three paragraphs should be merged as one.

Within red blood cells, lead reduces cell membrane flexibility and elevates the rate of erythrocyte breakdown, leading to anemia. Lead further specifically binds the delta-aminolevulinic acid dehydratase (ALAD) enzyme which is important in the heme biosynthetic pathway. This enzyme is involved in the condensation of glycine and succinyl CoA, and decarboxylation into delta-aminolevulinic acid (ALA) during the initial step of heme synthesis that takes place in the mitochondria before subsequent intermediate steps that take place in the cytoplasm and mitochondria again.

Porphobilinogen is formed by the combination of two molecules of δ-ALA with the help of the enzyme δ-aminolevulinic acid dehydratase (δ-ALAD) in the cytosol.

Subsequently, the enzyme ferrochelatase in the mitochondria facilitates the incorporation of a ferrous ion (Fe2+) into protoporphyrin IX to create heme. Delta-ALAD plays a vital role in lead poisoning, as its inhibition reduces heme production, leading to an increase in δ-ALA levels in the blood and urine of individuals exposed to lead. The synthesis of heme is not significantly affected until δ-ALAD activity is inhibited by 80–90%,¹⁸ which typically happens at a blood lead concentration of around 55 μg/dL.

Methods:
Throughout the manuscript, change μl and ml to μL and mL.

Results & Discussion:
For each correlation, an effect size for the correlations by adding the r2 value to each statistical calculation and add the column to reflect it. Then add this information into the discussion and update it accordingly.

Results:
Should report in the text the data presented in the table with the degrees of freedom as would be expected of any manuscript.

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report 09 Aug 2023

Lorenz S. Neuwirth, SUNY Neuroscience Research Institute, Old Westbury, New York, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.120325.r184451

In the abstract the language is unclear in the following:

Background: it should read "in sub-Saharan Africa the population of Uganda has experienced increased environmental exposures to lead as a contaminant that subsequently has caused elevated

In the abstract the language is unclear in the following:

Background: it should read "in sub-Saharan Africa the population of Uganda has experienced increased environmental exposures to lead as a contaminant that subsequently has caused elevated blood lead levels in children as a neurotoxicant within these areas." The sentences that follows..."Its levels in one's environment account for their blood lead levels."...is confusing and unclear. Eliminate and replace with a range of blood lead levels that were previously observed in this area prior to the population explosion and how it impacts the lives of children when exposed to a neurotoxin like lead.
Methods: A statement of how many children, their age range, and gender proportion should be included to help the reader obtain a sense of what this study did methodologically. Also, the measurements for BLL, Hb, and ALAD should be noted in parentheses following each term (i.e., BBL measured in ug/dL). The method of spectrophotometry should be changed to atomic absorption spectrophotometry with graphite furnace analysis.
Results: No indication of the mean and standard deviation of the BLLs and Hb by age and gender are noted, which is problematic. Moreover, the correlations that are noted which state a relationship is too general and needs to be more explicit. Indicate whether a positive linear, negative linear, non-linear, or curvilinear (i.e., U-shaped or inverted-U-shaped) relationships are observed for each comparisons. Further, the correlations should be additionally supported by an effect size using a Cohen's d or ds to fully allow the reader to understand the impacts of these findings and their potential generalization into work being done on a similar level in other countries.

Key words: I would suggest only keep Blood lead levels, then add d-aminolevulinc acid dehydratase enzyme, hemoglobin, childhood lead exposure,

Introduction: The writing has too many split infinitives and/or awkward phrasing and should be proof read to ensure clarity before submitting (i.e., first sentence..."numerous simultaneous transitions...).

Lead is an environmental contaminant at high- and low-exposure levels. However, levels that are deems low enough to be safe are still detrimental to the developing central nervous system in children as lead is a neurotoxin with no safe level of exposure. This should be somehow added in the first paragraph to establish a stronger problem statement. Also, this would avoid the ambiguities presented by stating lead is an accumulative toxin as this suggests low levels are safe and tolerable, which they are not.

The following sentence on the impacts of lead exposures in children should have clear blood lead levels noted as to which organ systems are disrupted at what lead exposure levels (i.e., low-levels reduce IQ, alter hormones, and stunt growth, but to not harm the lung, stomach, and etc.).

The sentence should be re-written..."Lead ion absorption in the intestines has been reported to exhibit a negative linear relationship between increased intestinal absorption and decreased hemoglobin (Hb) levels." This sentence also needs an in-text citation to support its claim.

Please define and/or elaborate..."lead sinks in read blood cells"... do you mean its ability to absorb into and bind?

The sentence that "ALA triggers the production of ROS..." should be followed by another sentence on the functional significance in why this pathway is activated. The increased lead body/brain burden is activating oxidative stress and ALA is mobilized as a defensive or counteractive mechanisms to trigger ROS to try to protect the brain and body from lead (neuro)toxic exposures at the physiological levels.

Overall, the introduction needs to be tighter, better framed, and more information on the clinical impacts of children. Additionally, from where exactly are they obtaining such exposures? A few sentences should address this issue. Then when are children identified to be lead poisoned (i.e., birth, age 1-5, once they start school, or when emergency hospitalization is required). This needs to be clear for the reader as different countries may have different proactive measures or reactive measures for treating children for lead poisoning in clinics. The pharmacokinetics for how long the children are exposed set up a government and policy susceptibility measure for these children that appears to be overlooked and would be important to include here. Identifying biomarkers (ALAD polymorphisms) and BLLs are measures of the lead insult already harming children, but at what ages are these biomarkers being reported and proactively assessed for in the population?

Additionally, the ALAD-1 and ALAD-2 allele should be explained in terms of their significance, what each allele is associated with in terms of medical or neurological risk factors or susceptibility factors for conditions (i.e., including lead poisoning), and how other populations prevalence are similar and different from Uganda.

Methods Section:
How long did the transportation of samples take (i.e, hours)? And were they on dry or wet ice?
The lead standard concentrations should all be reported as it is unclear the step-wise increases used and this would help the reader understand the sensitivity range of the samples measured. What was the margin of error for the samples detection accuracy? This ought to be reported.
Note: ml and ul should be changed to mL and uL and made consistent throughout the manuscript.

Remove words like ours and we to prevent author bias throughout the manuscript. Write in the third person.

Data Analysis section: The statistical formulas used for each dataset should be described separately. Each test should include its alpha level for the significance threshold, the confidence interval, and the post hoc comparisons along with the effect sizes. Correlations should then be described after these methods to understand how the data were treated to draw the inferences that are reported in the manuscript.

Results section:
The results should be describe in clear statistical reporting formats including the degrees of freedom, and what each finding means. The way the statistics are reported is too brief and under informing and should not just point to the tables without further explanation.

In the Tables, the mean should be accompanied by the standard deviation, and the standard error of the mean. Also, the data should be broken down by males vs. females as well as combined genders as there are differences reported in the literature as a function of gender/sex.

It would be helpful to show a figure of the BLL differences as a a bar graph with means and standard error of the means for males, females, and combined gender and split into age groups dependent upon how the spread of the demographics sampled. Another figure should be similarly constructed on hemoglobin levels, and a third on the ALAD-1 and ALAD-2 molecular work. Then from here, the data should be explained and the rationale put forth as to why then correlations were employed and in what ways to explore which relationships. This would strengthen the manuscripts methodology significantly.

It would be helpful to include correlation scatterplots of each of the relationships noted for the reader to visualize and gain a sense of what was done and what the data offer as new information and findings.

Discussion section:

It is again too brief and does not link back to the original problem statement, the hypothesis under study, and the need for an improved understanding of ALAD-1 vs ALAD-2 in general then as it relates to lead poisoning. This should then be further compared to ALAD-1 and ALAD-2 in the literature, how lead poisoning may be both a risk for changes in ALAD genotype polymorphisms/alleles, and how lead poisoning may potentially compound or exacerbate ALAD-1 and ALAD-2 medical and neurological risk factors. It is plausible that multiple-generations of people living in lead exposed environments may become less or more susceptible to such exposures and epigenetic modifications of the ALAD alleles may be commensurate with such experiences. This should be approached and described in this section.

Lastly, what are the conclusions and takeaways for the reader? What should they be made more aware of publicly regarding both lead exposures in Uganda due to industrialization, urbanization, economic development, e-waste sites, etc. and how can an educational outreach of this information coupled with ALAD-1 and ALAD-2 genotype help to alert the public of their lived risks when residing in such an environment? This seems to be overlooked and would be important to add mean and purpose for the public regarding the work done. Also, what are the economic impacts of these 15.2% of children from the 7,000 inhabitants screened in this study over the lifespan? This would be a nice closure point to address and bring attention to the need for more research and support in this area.

References:
It seems to be too light for a manuscript of this type and I would suggest consulting the literature more and developing it to have at least 50-70 references.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

References

1. Cabañas E, Cruz G, Vasquez M, Joseph J, et al.: Dietary effects of lead as a neurotoxicant. 2023. 387-410 Publisher Full Text
2. Neuwirth LS, Emenike BU, Cruz GB, Cabañas E, et al.: Taurine-Derived Compounds Produce Anxiolytic Effects in Rats Following Developmental Lead Exposure.Adv Exp Med Biol. 2022; 1370: 445-460 PubMed Abstract | Publisher Full Text
3. Vasquez MA, Cruz GB, Cabañas E, Joseph JN, et al.: In Vivo Sex-Dependent Effects of Perinatal Pb2+ Exposure on Pilocarpine-Induced Seizure Susceptibility and Taurine Neuropharmacology.Adv Exp Med Biol. 2022; 1370: 481-496 PubMed Abstract | Publisher Full Text
4. Cruz GB, Vasquez MA, Cabañas E, Joseph JN, et al.: Developmental Lead Exposure in Rats Causes Sex-Dependent Changes in Neurobiological and Anxiety-Like Behaviors that Are Improved by Taurine Co-treatment.Adv Exp Med Biol. 2022; 1370: 461-479 PubMed Abstract | Publisher Full Text
5. Neuwirth LS, Cabañas E, Cadet P, Zhu W, et al.: Cereal and Juice, Lead and Arsenic, Our Children at Risk: A Call for the FDA to Re-Evaluate the Allowable Limits of Lead and Arsenic That Children May Ingest.Int J Environ Res Public Health. 2022; 19 (10). PubMed Abstract | Publisher Full Text
6. Neuwirth LS, Lopez OE, Schneider JS, Markowitz ME: Low-level Lead Exposure Impairs Fronto-executive Functions: A Call to Update the DSM-5 with Lead Poisoning as a Neurodevelopmental Disorder.Psychol Neurosci. 2020; 13 (3): 299-325 PubMed Abstract | Publisher Full Text
7. Neuwirth LS: Resurgent lead poisoning and renewed public attention towards environmental social justice issues: A review of current efforts and call to revitalize primary and secondary lead poisoning prevention for pregnant women, lactating mothers, and children within the U.S.Int J Occup Environ Health. 2018; 24 (3-4): 86-100 PubMed Abstract | Publisher Full Text

Competing Interests: I have indicated some of my own papers for the authors to consider, not just as potential references, but as to how they could frame their introductions on the topic they present more clearly. This is done in full transparency in the hopes that these authors can improve their work to meet the requirements of publication so that the children of Uganda can have a good starting point for addressing these lead exposures that they face.

Reviewer Expertise: Developmental behavioral neurotoxicology of lead exposures with over 20 years experience in basic research/pre-clinical models of childhood lead expsosures.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Reviewer Report 09 Jun 2022

Muhammad Sajid Hamid Akash, Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Government College University, Faisalabad, Pakistan

Approved with Reservations

https://doi.org/10.5256/f1000research.120325.r138566

This manuscript describes in detail the role of ALAD (delta-aminolevulinic acid dehydratase) in enzymatic activity and its susceptibility to lead toxicity in urban children of Uganda. This study seems interesting, but there are certain flaws and shortcomings that need careful ... Continue reading

This manuscript describes in detail the role of ALAD (delta-aminolevulinic acid dehydratase) in enzymatic activity and its susceptibility to lead toxicity in urban children of Uganda. This study seems interesting, but there are certain flaws and shortcomings that need careful attention of the authors to revise their manuscript on the basis of the following comments:

What are the toxic values of lead? Give the ratio of exposure to lead globally as well as in Uganda. Furthermore, introduction section of this article needs some major revisions to add more data about the role of exposure of lead on various enzymes notably delta aminolevulinic acid by considering the latest studies conducted on the role of lead on ALAD and various other metabolizing enzymes. Following references (Environ Sci Pollut Res. 2022; https://doi.org/10.1007/s11356-022-20069-5 and Environ Sci Pollut Res. 2021; https://doi.org/10.1007/s11356-021-15323-1) may help the authors in this regards.
Formatting, spacing and grammatical mistakes should be corrected.
Have you taken socio-demographic information of the patients?
What were the inclusion and exclusion criteria of your study?
In result section, a table related to susceptibility (increased or decreased) population is missing. Add this data as your title and aim is related to susceptibility also.
Discuss your findings with the recent previous studies under the discussion section, only 7 references are supporting your discussion, of whom no study was related to your findings.
What are the limitations of your study?
Conclude your findings comprehensively under a separate heading.
Citations are missing in most of the sentences of introduction (especially first 3 to 4 sentences). Try to add most recent citations.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

References

1. Qader A, Rehman K, Akash MSH: Biochemical profiling of lead-intoxicated impaired lipid metabolism and its amelioration using plant-based bioactive compound.Environ Sci Pollut Res Int. 2022. PubMed Abstract | Publisher Full Text
2. Qader A, Rehman K, Akash M: Genetic susceptibility of δ-ALAD associated with lead (Pb) intoxication: sources of exposure, preventive measures, and treatment interventions. Environmental Science and Pollution Research. 2021; 28 (33): 44818-44832 Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: EDCs-induced cardiometabolic disorders; Biochemical Genetics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Muhammad Sajid Hamid Akash, Government College University, Faisalabad, Pakistan
Lorenz S. Neuwirth, SUNY Neuroscience Research Institute, Old Westbury, USA
Howard Hu, University of Southern California, Los Angeles, USA

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Reviewer Report

33 Views

11 Nov 2024 | for Version 2

Howard Hu, Department of Population and Public Health Sciences, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

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Not Approved

The subjects of this report are novel and important, in that lead exposure remains one of the top environmental health problems in the world, whereas studies of lead exposure (with blood lead as a biomarker of exposure) in Africa remain sparse. Moreover, the addition of analyses of the results across ALAD genotype, one of the most important known genetic modifiers of lead toxicokinetics, is quite novel. The overall manuscript and reporting are quite good, BUT there are several issues, mostly reflecting a lack of analyses and reporting format that align with epidemiology (and this is, essentially, an epidemiological study), some inconsistencies in the data, and some further improvements that are needed in terms of the writing. Details are provided below.
ABSTRACT
- 2^nd sentence: “Mean blood levels of 332 ug/dL, 120 ug/dL…”.
  - I would not call a mean blood lead level of 332 or 120 ug/dL as “elevated blood lead levels”---I think these should be identified as a examples of extreme clinical lead poisoning, whereas the mean blood lead levels of 25, 11, and 10 ug/dL are examples of elevated subclinical lead exposures.
  - The sentence itself is not a full sentence (no verb!).
- The methods section should include a few words describing the population sampled, e.g., “randomly selected children residing in the Katanga slum of Kampala, Uganda”.”
- The results (see discussion of results below)
INTRO
- In general, the discussion of lead’s molecular impacts could be shortened, if space is a limitation
- Last para: ALAD polymorphism is only one of several polymorphisms that have been associated with altered lead metabolism and/or susceptibility to toxic effects (examples of others: the VDR, HFE, Dopamine metabolism, etc.).
METHODS
- In general, excellent description of lab methods (which could be shortened if more space is needed)
- From epidemiological perspective: The authors should clearly state the number of individuals studied. It says “Duplicate samples of venous blood (5 mg; n=198) were drawn from each study participants…” but that that suggests the actual number of individuals was n=198, each having duplicate samples; or n=198 samples/2 = 99 individuals, each having duplicate samples. Re-word to say “Duplicate samples of venous blood (5 mg) were drawn from each study participants (n=198)…”
RESULTS
- From an epidemiological perspective, it is important to go over basic demographics (age, sex), data for which should be reported in the text as well as added to a revised Table 1. Given that there is only 1 individual with the ALAD 2-2 genotype, I would advise reporting this individual’s values in the text, but in the Table, merging this individual’s data with the ALAD 1-2 data and simply calling this group “ALAD 1-2 and 2-2” (it makes no sense statistically to analyze this one individual’s data as a separate category).
- I suggest that the variables are displayed in rows (not columns), the columns can take the form of “ALAD1-1”, “ALAD1-2 and 2-2”, and “Total”, and rows can be added at the top to report data on n (number of individuals in each group”, sex (Male v. Female); mean, SD, range of ages. .
- 99.5% of individuals have the ALAD1 allele (exclusively?); whereas 0.11% (NOT 0.05%) carried the ALAD2 allele. This also has to be corrected in the abstract
- The hematocrit results do not make sense. Hemoglobin and hematocrit level typically are highly correlated, whereas comparing the ALAD 1-1 with the ALAD 1-2 groups, the hemoglobin levels are lower whereas the hematocrit levels are higher, respectively. Such a discrepancy can sometimes occur, but only under unusual circumstance, such as if many of the individuals in the study had a hemoglobinopathy, such as Thalassemia, by which the hemoglobin levels are low, and the body tries to compensate by producing more red cells, which increases the hematocrit. The authors should either check their data to see if perhaps the values have been switched; or determine if hemoglobinopathy could be a major factor in this group of individuals, etc. Either way, they need to address this discrepancy in the discussion.
- Table 2: I would not analyze or describe these relationships as “correlations” with r-values. The differences in blood lead, ALAD enzyme activity, hemoglobin levels, and hematocrit across categories of ALAD genotype should be analyzed and reported as the results of ANOVA. In addition, all such results could easily be merged into Table 1.
- Regarding the data, I think there is a missed opportunity to use multivariate regressions to examine inter-relationships, such as treating hemoglobin levels or hematocrit levels as an outcome in relationship to (a) a model with age, sex, ALAD-1/1 or ½ versus wildtype (ALAD-1/1) as independent variables; and (b) a second model with age, sex, ALAD enzyme activity as independent variables.
DISCUSSION
- It would be more accurate to say that the 0.5% prevalence of ALAD2 is in agreement with other data specific to individuals in Africa, i.e., the study of Liberians by Benkmann et al (ref. 41).
- Consider adding discussion of the potential sources of the elevated lead exposure among these individuals living in this neighborhood of Kampala, particularly since lead exposure in low- and middle-income countries has recently become the topic of intense interest and activity by government agencies (such as UNICEF, US Agency for International Development, the World Bank) and NGO’s (such as Pure Earth).

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Environmental epidemiology, internal medicine, occupational/environmental medicine, toxicology.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Reviewer Report

6 Views

14 Jun 2024 | for Version 2

Lorenz S. Neuwirth, SUNY Neuroscience Research Institute, Old Westbury, New York, USA

6 Views Cite this report Responses(0)

Approved

Abstract:
In the abstract, the conclusion should contain more information containing a discussion of the results and then the conclusion. As it reads, it is incomplete.

Introduction:
It may just be the formatting of how the manuscript is set up, but the following three paragraphs should be merged as one.

Within red blood cells, lead reduces cell membrane flexibility and elevates the rate of erythrocyte breakdown, leading to anemia. Lead further specifically binds the delta-aminolevulinic acid dehydratase (ALAD) enzyme which is important in the heme biosynthetic pathway. This enzyme is involved in the condensation of glycine and succinyl CoA, and decarboxylation into delta-aminolevulinic acid (ALA) during the initial step of heme synthesis that takes place in the mitochondria before subsequent intermediate steps that take place in the cytoplasm and mitochondria again.

Porphobilinogen is formed by the combination of two molecules of δ-ALA with the help of the enzyme δ-aminolevulinic acid dehydratase (δ-ALAD) in the cytosol.

Subsequently, the enzyme ferrochelatase in the mitochondria facilitates the incorporation of a ferrous ion (Fe2+) into protoporphyrin IX to create heme. Delta-ALAD plays a vital role in lead poisoning, as its inhibition reduces heme production, leading to an increase in δ-ALA levels in the blood and urine of individuals exposed to lead. The synthesis of heme is not significantly affected until δ-ALAD activity is inhibited by 80–90%,¹⁸ which typically happens at a blood lead concentration of around 55 μg/dL.

Methods:
Throughout the manuscript, change μl and ml to μL and mL.

Results & Discussion:
For each correlation, an effect size for the correlations by adding the r2 value to each statistical calculation and add the column to reflect it. Then add this information into the discussion and update it accordingly.

Results:
Should report in the text the data presented in the table with the degrees of freedom as would be expected of any manuscript.

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report

10 Views

09 Aug 2023 | for Version 1

Lorenz S. Neuwirth, SUNY Neuroscience Research Institute, Old Westbury, New York, USA

10 Views Cite this report Responses(0)

Approved With Reservations

In the abstract the language is unclear in the following:

Background: it should read "in sub-Saharan Africa the population of Uganda has experienced increased environmental exposures to lead as a contaminant that subsequently has caused elevated blood lead levels in children as a neurotoxicant within these areas." The sentences that follows..."Its levels in one's environment account for their blood lead levels."...is confusing and unclear. Eliminate and replace with a range of blood lead levels that were previously observed in this area prior to the population explosion and how it impacts the lives of children when exposed to a neurotoxin like lead.
Methods: A statement of how many children, their age range, and gender proportion should be included to help the reader obtain a sense of what this study did methodologically. Also, the measurements for BLL, Hb, and ALAD should be noted in parentheses following each term (i.e., BBL measured in ug/dL). The method of spectrophotometry should be changed to atomic absorption spectrophotometry with graphite furnace analysis.
Results: No indication of the mean and standard deviation of the BLLs and Hb by age and gender are noted, which is problematic. Moreover, the correlations that are noted which state a relationship is too general and needs to be more explicit. Indicate whether a positive linear, negative linear, non-linear, or curvilinear (i.e., U-shaped or inverted-U-shaped) relationships are observed for each comparisons. Further, the correlations should be additionally supported by an effect size using a Cohen's d or ds to fully allow the reader to understand the impacts of these findings and their potential generalization into work being done on a similar level in other countries.

Key words: I would suggest only keep Blood lead levels, then add d-aminolevulinc acid dehydratase enzyme, hemoglobin, childhood lead exposure,

Introduction: The writing has too many split infinitives and/or awkward phrasing and should be proof read to ensure clarity before submitting (i.e., first sentence..."numerous simultaneous transitions...).

Lead is an environmental contaminant at high- and low-exposure levels. However, levels that are deems low enough to be safe are still detrimental to the developing central nervous system in children as lead is a neurotoxin with no safe level of exposure. This should be somehow added in the first paragraph to establish a stronger problem statement. Also, this would avoid the ambiguities presented by stating lead is an accumulative toxin as this suggests low levels are safe and tolerable, which they are not.

The following sentence on the impacts of lead exposures in children should have clear blood lead levels noted as to which organ systems are disrupted at what lead exposure levels (i.e., low-levels reduce IQ, alter hormones, and stunt growth, but to not harm the lung, stomach, and etc.).

The sentence should be re-written..."Lead ion absorption in the intestines has been reported to exhibit a negative linear relationship between increased intestinal absorption and decreased hemoglobin (Hb) levels." This sentence also needs an in-text citation to support its claim.

Please define and/or elaborate..."lead sinks in read blood cells"... do you mean its ability to absorb into and bind?

The sentence that "ALA triggers the production of ROS..." should be followed by another sentence on the functional significance in why this pathway is activated. The increased lead body/brain burden is activating oxidative stress and ALA is mobilized as a defensive or counteractive mechanisms to trigger ROS to try to protect the brain and body from lead (neuro)toxic exposures at the physiological levels.

Overall, the introduction needs to be tighter, better framed, and more information on the clinical impacts of children. Additionally, from where exactly are they obtaining such exposures? A few sentences should address this issue. Then when are children identified to be lead poisoned (i.e., birth, age 1-5, once they start school, or when emergency hospitalization is required). This needs to be clear for the reader as different countries may have different proactive measures or reactive measures for treating children for lead poisoning in clinics. The pharmacokinetics for how long the children are exposed set up a government and policy susceptibility measure for these children that appears to be overlooked and would be important to include here. Identifying biomarkers (ALAD polymorphisms) and BLLs are measures of the lead insult already harming children, but at what ages are these biomarkers being reported and proactively assessed for in the population?

Additionally, the ALAD-1 and ALAD-2 allele should be explained in terms of their significance, what each allele is associated with in terms of medical or neurological risk factors or susceptibility factors for conditions (i.e., including lead poisoning), and how other populations prevalence are similar and different from Uganda.

Methods Section:
How long did the transportation of samples take (i.e, hours)? And were they on dry or wet ice?
The lead standard concentrations should all be reported as it is unclear the step-wise increases used and this would help the reader understand the sensitivity range of the samples measured. What was the margin of error for the samples detection accuracy? This ought to be reported.
Note: ml and ul should be changed to mL and uL and made consistent throughout the manuscript.

Remove words like ours and we to prevent author bias throughout the manuscript. Write in the third person.

Data Analysis section: The statistical formulas used for each dataset should be described separately. Each test should include its alpha level for the significance threshold, the confidence interval, and the post hoc comparisons along with the effect sizes. Correlations should then be described after these methods to understand how the data were treated to draw the inferences that are reported in the manuscript.

Results section:
The results should be describe in clear statistical reporting formats including the degrees of freedom, and what each finding means. The way the statistics are reported is too brief and under informing and should not just point to the tables without further explanation.

In the Tables, the mean should be accompanied by the standard deviation, and the standard error of the mean. Also, the data should be broken down by males vs. females as well as combined genders as there are differences reported in the literature as a function of gender/sex.

It would be helpful to show a figure of the BLL differences as a a bar graph with means and standard error of the means for males, females, and combined gender and split into age groups dependent upon how the spread of the demographics sampled. Another figure should be similarly constructed on hemoglobin levels, and a third on the ALAD-1 and ALAD-2 molecular work. Then from here, the data should be explained and the rationale put forth as to why then correlations were employed and in what ways to explore which relationships. This would strengthen the manuscripts methodology significantly.

It would be helpful to include correlation scatterplots of each of the relationships noted for the reader to visualize and gain a sense of what was done and what the data offer as new information and findings.

Discussion section:

It is again too brief and does not link back to the original problem statement, the hypothesis under study, and the need for an improved understanding of ALAD-1 vs ALAD-2 in general then as it relates to lead poisoning. This should then be further compared to ALAD-1 and ALAD-2 in the literature, how lead poisoning may be both a risk for changes in ALAD genotype polymorphisms/alleles, and how lead poisoning may potentially compound or exacerbate ALAD-1 and ALAD-2 medical and neurological risk factors. It is plausible that multiple-generations of people living in lead exposed environments may become less or more susceptible to such exposures and epigenetic modifications of the ALAD alleles may be commensurate with such experiences. This should be approached and described in this section.

Lastly, what are the conclusions and takeaways for the reader? What should they be made more aware of publicly regarding both lead exposures in Uganda due to industrialization, urbanization, economic development, e-waste sites, etc. and how can an educational outreach of this information coupled with ALAD-1 and ALAD-2 genotype help to alert the public of their lived risks when residing in such an environment? This seems to be overlooked and would be important to add mean and purpose for the public regarding the work done. Also, what are the economic impacts of these 15.2% of children from the 7,000 inhabitants screened in this study over the lifespan? This would be a nice closure point to address and bring attention to the need for more research and support in this area.

References:
It seems to be too light for a manuscript of this type and I would suggest consulting the literature more and developing it to have at least 50-70 references.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

References

1. Cabañas E, Cruz G, Vasquez M, Joseph J, et al.: Dietary effects of lead as a neurotoxicant. 2023. 387-410 Publisher Full Text
2. Neuwirth LS, Emenike BU, Cruz GB, Cabañas E, et al.: Taurine-Derived Compounds Produce Anxiolytic Effects in Rats Following Developmental Lead Exposure.Adv Exp Med Biol. 2022; 1370: 445-460 PubMed Abstract | Publisher Full Text
3. Vasquez MA, Cruz GB, Cabañas E, Joseph JN, et al.: In Vivo Sex-Dependent Effects of Perinatal Pb2+ Exposure on Pilocarpine-Induced Seizure Susceptibility and Taurine Neuropharmacology.Adv Exp Med Biol. 2022; 1370: 481-496 PubMed Abstract | Publisher Full Text
4. Cruz GB, Vasquez MA, Cabañas E, Joseph JN, et al.: Developmental Lead Exposure in Rats Causes Sex-Dependent Changes in Neurobiological and Anxiety-Like Behaviors that Are Improved by Taurine Co-treatment.Adv Exp Med Biol. 2022; 1370: 461-479 PubMed Abstract | Publisher Full Text
5. Neuwirth LS, Cabañas E, Cadet P, Zhu W, et al.: Cereal and Juice, Lead and Arsenic, Our Children at Risk: A Call for the FDA to Re-Evaluate the Allowable Limits of Lead and Arsenic That Children May Ingest.Int J Environ Res Public Health. 2022; 19 (10). PubMed Abstract | Publisher Full Text
6. Neuwirth LS, Lopez OE, Schneider JS, Markowitz ME: Low-level Lead Exposure Impairs Fronto-executive Functions: A Call to Update the DSM-5 with Lead Poisoning as a Neurodevelopmental Disorder.Psychol Neurosci. 2020; 13 (3): 299-325 PubMed Abstract | Publisher Full Text
7. Neuwirth LS: Resurgent lead poisoning and renewed public attention towards environmental social justice issues: A review of current efforts and call to revitalize primary and secondary lead poisoning prevention for pregnant women, lactating mothers, and children within the U.S.Int J Occup Environ Health. 2018; 24 (3-4): 86-100 PubMed Abstract | Publisher Full Text

Competing Interests

I have indicated some of my own papers for the authors to consider, not just as potential references, but as to how they could frame their introductions on the topic they present more clearly. This is done in full transparency in the hopes that these authors can improve their work to meet the requirements of publication so that the children of Uganda can have a good starting point for addressing these lead exposures that they face.

Reviewer Expertise

Developmental behavioral neurotoxicology of lead exposures with over 20 years experience in basic research/pre-clinical models of childhood lead expsosures.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Reviewer Report

18 Views

09 Jun 2022 | for Version 1

Muhammad Sajid Hamid Akash, Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Government College University, Faisalabad, Pakistan

18 Views Cite this report Responses(0)

Approved With Reservations

This manuscript describes in detail the role of ALAD (delta-aminolevulinic acid dehydratase) in enzymatic activity and its susceptibility to lead toxicity in urban children of Uganda. This study seems interesting, but there are certain flaws and shortcomings that need careful attention of the authors to revise their manuscript on the basis of the following comments:

What are the toxic values of lead? Give the ratio of exposure to lead globally as well as in Uganda. Furthermore, introduction section of this article needs some major revisions to add more data about the role of exposure of lead on various enzymes notably delta aminolevulinic acid by considering the latest studies conducted on the role of lead on ALAD and various other metabolizing enzymes. Following references (Environ Sci Pollut Res. 2022; https://doi.org/10.1007/s11356-022-20069-5 and Environ Sci Pollut Res. 2021; https://doi.org/10.1007/s11356-021-15323-1) may help the authors in this regards.
Formatting, spacing and grammatical mistakes should be corrected.
Have you taken socio-demographic information of the patients?
What were the inclusion and exclusion criteria of your study?
In result section, a table related to susceptibility (increased or decreased) population is missing. Add this data as your title and aim is related to susceptibility also.
Discuss your findings with the recent previous studies under the discussion section, only 7 references are supporting your discussion, of whom no study was related to your findings.
What are the limitations of your study?
Conclude your findings comprehensively under a separate heading.
Citations are missing in most of the sentences of introduction (especially first 3 to 4 sentences). Try to add most recent citations.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

References

1. Qader A, Rehman K, Akash MSH: Biochemical profiling of lead-intoxicated impaired lipid metabolism and its amelioration using plant-based bioactive compound.Environ Sci Pollut Res Int. 2022. PubMed Abstract | Publisher Full Text
2. Qader A, Rehman K, Akash M: Genetic susceptibility of δ-ALAD associated with lead (Pb) intoxication: sources of exposure, preventive measures, and treatment interventions. Environmental Science and Pollution Research. 2021; 28 (33): 44818-44832 Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

EDCs-induced cardiometabolic disorders; Biochemical Genetics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Respond to this report

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Delta-Aminolevulinic acid dehydratase enzyme activity and susceptibility to lead toxicity in Uganda’s urban children

Abstract

Background

Methods

Results

Conclusions

Keywords

Revised Amendments from Version 1

Introduction

Methods

Ethical considerations

The study design

Assay for blood lead using atomic absorption spectrophotometer

Colorimetric determination of hemoglobin levels by blood cyanmethemoglobin reaction method

Determination of hematocrit levels of the study blood samples

Determination of delta-aminolevulinic acid dehydratase (ALAD) enzyme activity

Delta-aminolevulinic acid dehydratase (ALAD) genotyping

Data analysis

Results

Table 1.

Table 2.

Discussion

Conclusion

Study limitation

Data availability

Underlying data

Extended data

References

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