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\begin{document}

\title{Vitamin B12 Deficiency}



\author[1]{Janti Qar}%
\author[1]{Laila Azzam}%
\author[1]{Bahaa Al-Trad}%
\author[2]{Ayman AlZu'bi}%
\author[2]{Manal Abualarja}%
\author[1]{Khalid Al-Batayneh}%
\author[2]{Mazhar Al Zoubi}%
\affil[1]{Yarmouk University Faculty of Sciences}%
\affil[2]{Yarmouk University}%


\vspace{-1em}



  
  \date{May 04, 2024}


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\selectlanguage{english}
\begin{abstract}
Objectives: Vitamin B12 deficiency has been associated with certain
serious disorders such as megaloblastic anemia as well as
cardiovascular, neurological, and psychological disorders. Vitamin B12
deficiency is a worldwide health concern and a severe health problem in
certain developing countries. In the current study, we aimed to evaluate
the situation of vitamin B12 and estimate the prevalence of vitamin B12
deficiency in southern Jordan. Methods: The study population included
281 participants from Aqaba city: 71.2\% females and 28.8\% males with a
mean age of 30.01\selectlanguage{ngerman}±13.5. Results: The results showed that the average
vitamin B12 level was 277.34±103.70 pg/mL. The study population showed
that 69 (24.6\%) participants had a vitamin B12 deficiency. The current
findings showed a lower frequency of vitamin B12 deficiency in the
studied population compared to the rest of Jordan, especially in the
north. The females reported a significantly higher frequency of vitamin
B12 deficiency (29.0\%) compared to the male group (13.6\%). In
addition, the CBC parameters showed a significant association between
the level of vitamin B12 and Hb, RBC, PCV, and platelet count with a
p-value = 0.044, \textless{}0.001, 0.021, 0.025, respectively. On the
other hand, the results showed a lack of association between the vitamin
B12 level and MCV and age group. Conclusion: In conclusion, the current
study supports our previous findings regarding the correlation between
geographical locations and the deficiency of vitamin B12. These findings
can be attributed to the lifestyle and nutrient situation in Aqaba city
which relies more on seafood. Moreover, other factors such as genetic
predisposition can be excluded as an association factor. Therefore, more
studies are required to evaluate vitamin B12 levels in Jordan at a
higher scale that will include genetic analysis as well as food habits.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Introduction}

Vitamin B12 (Cobalamin; Cbl) is a water-soluble molecule characterized
by its chemical complexity, large size, and cobalt ion which is
centrally located in the corrin ring\textsuperscript{1}. Vitamin B12 is
essentially obtained from dietary sources such as red meat, dairy
products, eggs, and fish\textsuperscript{2-4}. Absorption and
transportation of vitamin B12 are processes that occur by hydrophobic
binding protein carriers including haptocorrin (HC), intrinsic factor
(IF), and transcobalamin (TCN)\textsuperscript{5-7}. Around 80\% of
cobalamin is carried by inactive haptocorrin (or transcobalamin I) and
20\% is transported by active TCN II in the
bloodstream\textsuperscript{8-10}. The deficiency of vitamin B12 is
attributed to many factors including insufficient dietary supply,
malabsorption, and deficient absorption cases\textsuperscript{11-15}. In
addition, many risk factors are related to vitamin B12 deficiency
including genetic predisposition, age, weight, gender, and ethnic
variations\textsuperscript{16-19}.

Vitamin B12 has a vital role in many processes including the synthesis
of nucleic acids, regulating the metabolism of the macronutrients
(proteins, fats, and carbohydrates), as well as some neurological
functions \textsuperscript{20-24}. Moreover, vitamin B12 plays a
necessary role in myelin biosynthesis, manufacturing, and development of
erythrocytes (hematopoiesis)\textsuperscript{11,25,26}. Therefore,
vitamin B12 deficiency is associated with serious neurological,
hematological, and metabolic disorders\textsuperscript{21,27-30}.
Vitamin B12 deficiency is most prevalent in the elderly, children,
pregnant women, nursing mothers, and vegetarians\textsuperscript{31-34}.
The most significant diagnostic characteristics and symptoms related to
a deficiency of B-12 are fatigue, memory impairment, skin pallor, skin
hyperpigmentation, glossitis, and severe hematological, neurological,
and psychiatric disorders\textsuperscript{35-41}.

Vitamin B12 deficiency is a worldwide concern\textsuperscript{16,42,43}.
Notably, vitamin B12 deficiency is not prevalent in wealthy countries
except in the elderly, compared to developing countries where the
prevalence of vitamin B12 deficiency can reach more than 50\% of the
population\textsuperscript{16,42,44-49}. For instance, the prevalence of
vitamin B12 deficiency in the United States and the United Kingdom is
approximately 6\% in people under the age of 60 and about 20\% of those
over 60 years, whereas countries in Latin America have a deficiency
incidence of approximately 40\%\textsuperscript{50-56}. In Jordan,
vitamin B12 deficiency is a considerable health
problem\textsuperscript{57}, where the prevalence varies according to
the study population in the last decades\textsuperscript{57-59}. In
different studies in Jordan, vitamin B12 deficiency was estimated at
different prevalence (16--50\%) with the highest prevalence in the
elderly above 55 years old\textsuperscript{16,17,59-61}.

Based on our previous findings, we proposed the geographical location as
an important factor in the estimation of vitamin B12 which may be
related to socioeconomic status, lifestyle, and cultural background. We
reported significant differences in the frequencies of vitamin B12
deficiency in different locations in Jordan. In particular, the results
showed a lower frequency of vitamin B12 deficiency in the southern parts
of Jordan compared to the northern cities\textsuperscript{17}.
Consequently, the current study aimed to evaluate vitamin B12 levels
among the Jordanian population in Aqaba city (the most southern city)
and to assess the prevalence of vitamin B12 deficiency in Aqaba's
Jordanian population. This study will complete the portrait of the
national situation of vitamin B12 level.

\textbf{Materials and Methods}

\textbf{Study Design}

This retrospective study was performed in the most southern part of
Jordan (Aqaba province). Venous blood samples were collected from
different hospitals in Aqaba, with a total number of 281 samples.
Written informed consent was obtained from all participants, and the
data were collected where it related to location, gender, age, and
general health of the participants who were living in Aqaba. Exclusion
criteria included subjects who showed diseases which lead to cobalamin
deficiency such as peptic ulcer, gastrointestinal problem, pregnancy,
diabetes, and individuals under vitamin B12 supplementation.

\textbf{Blood Sampling}

Blood samples were collected from hospitals in the Aqaba governorate
then serum was preserved at -70\selectlanguage{ngerman}°C until use to measure the level of
vitamin B12 and further study according to standard procedures using
commercially available kits. Laboratory analyses were performed at
hospital laboratories in Aqaba, Jordan. The level of vitamin B12 was
assessed based on sandwich enzyme-linked immunosorbent assay technology,
according to the manufacturer's protocol (Cyanocobalamin (Vitamin B12)
ELISA Kit, Abbexa, UK).

\textbf{Statistical analysis}

Prevalence of Vitamin B12 deficiency was evaluated in Aqaba province
where all the statistical analysis was done using SPSS software version
25. Categorical data are expressed in frequency and percentages, scale
data are expressed in mean ±SD, chi-square test of independence was used
to examine the association between categorical data. Pearson\selectlanguage{english}'s
correlation was used to analyze the correlation between scale data, an
independent t-test was used for B12 mean differences between both
genders, \emph{P} -values \textless{} 0.05 were considered statistically
significant.

\textbf{Results}

\textbf{Demographic characteristics of the study population}

The study recruited 281 participants from Aqaba city; including 200
females (71.2 \%) and 81 males (28.8 \%). The ages ranged between 0 and
80 years with a mean age of 30.01+-13.5 (31.9+-16.49 for males and
29.3+-12.05 for females), where the age group between 0-39 years was more
prominent 217(77.2\%) as compared to the age group \selectlanguage{english}[?]40 years 64(22.8\%).
The mean of B12 for the study population was 277.34+-103.70 pg/mL.

The cutoff value of vitamin B12 level was set at 200 pg/mL, where the
individuals with vitamin B12 level less than 200 pg/mL were categorized
as deficient. Based on this categorization the results showed that 212
(75.4\%) of participants had a normal B12 level while 69 (24.6\%) of
them had a B12 deficiency. According to the gender groups, the females
reported a significantly higher frequency of vitamin B12 deficiency
(29.0\%) compared to the male group (13.6\%). In addition, the mean of
Hb level for participants was 12.66+-1.78 whereas the mean of RBC for
participants was 4.54+-0.50 as shown in table 1.

\textbf{Mean differences in vitamin B12 levels between males and
females.}

The mean differences of serum B12 level between males and females were
determined by an independent sample t-test and the results are shown in
Table 2. Our study showed a statistically significant difference in the
mean of serum B12 between genders (\emph{P} -value = 0.002), where the
mean of serum B12 in females was 265.09 +-106.35 pg/mL while it was
307.59 +-90.75 pg/mL in males.

In addition, a Chi-Square independence test was used to examine the
association between B12 deficiency and gender. The findings showed that
there was a statistically significant association between gender and B12
deficiency (X\textsuperscript{2} = 7.399, (\emph{P} -value = 0.007))
which indicates that the females had a higher proportion of B12
deficiency compared to the males as shown in table 3 and figure 1.

\textbf{Association between age group and vitamin B12 level.}

The current study reported a lack of association between the B12
deficiency and age groups (\textless{} 40, \selectlanguage{english}[?] 40 years) according to the
chi-square test (X\textsuperscript{2} = 2.428, (\emph{P} -value =
0.119)) as illustrated in table 4. An independent sample t-test was also
used to test the mean differences of B12 concerning age groups and the
results showed no statistically meaningful differences between them
(\emph{P} =0.661) as shown in table 5.

\textbf{Association between CBC parameters and serum vitamin B12}

The association between vitamin B12 and CBC parameters was analyzed
using Pearson's correlation where our results revealed that there was a
statistically significant correlation between Hb level and vitamin B12
level as shown in table 6. In addition, a statistically significant
correlation was observed between RBC and vitamin B12 level (r=
0.311,\emph{P} \textless{}0.001). Pearson product-moment showed there
was a statistically significant positive correlation between serum B12
and PCV (R=0.192, \emph{P} =0.021), B12 serum and PLT (R=0.193, \emph{P}
=0.025) while no statistically significant correlation was found with
MCV (R= - 0.080, \emph{P} =0.354) as illustrated in table 6.

\textbf{The relationship between geographical location and vitamin B12
levels.}

The results of this study support the previous findings for the four
regions in Jordan; Irbid, Karak, Tafila, and Ma'an, where the
percentages of B12 deficiency in these regions were 41\%, 35\%, 27\% and
26\% respectively \textsuperscript{57}. The current study evaluated B12
deficiency in Aqaba, which was found to be 24.6\%. Figures 2 and 3
illustrate a comparison between the frequencies of vitamin B12
deficiencies in five regions that cover Jordan from north to south.

\textbf{Discussion}

In the Middle East, different countries have reported a high prevalence
of vitamin B12 deficiency that is resulted in pernicious anemia
{[}94{]}. Similarly, the deficiency of vitamin B12 in Jordan is a health
concern that needs to be considered seriously. Previous studies showed
differences in the prevalence of vitamin B12 deficiency in Jordan. For
instance, in a study that included two different populations, the
researchers reported 50.8\% of vitamin B12 deficiency among the Arab
population and 46.9\% among Circassians\textsuperscript{62}. Other
studies reported 10\% \textsuperscript{58}, 24\%\textsuperscript{57},
27\%\textsuperscript{63}, 30\%\textsuperscript{16} and
44.6\%\textsuperscript{64} of vitamin B12 deficiency. The variation in
the prevalence can be attributed to the differences between the study
populations and age groups\textsuperscript{16,57-61}.

In a recent study, it was shown that vitamin B12 deficiency varies in
different provinces of Jordan; Irbid, Karak, Tafilah, and
Ma\selectlanguage{english}'an\textsuperscript{57}. In this retrospective study, we evaluated
vitamin B12 levels in a Jordanian population in Aqaba city and
correlated it with gender, age, and CBC parameters. The current study
reported a 24.6 \% vitamin B12 deficiency in the Aqaba province
population. These findings support our previous report detailing the
trend of vitamin B12 deficiency according to the geographical location
in Jordan. Additionally, the results reported a significant difference
between the means of vitamin B12 levels in males and females, 307.59
+-90.75, 265.09 +-106.35, respectively (\emph{p} = 0.002). In particular,
females showed a higher frequency of vitamin B12 deficiency (29\%)
compared to the male group (14\%). Inconsistently with a previous study,
the researchers showed that males had a higher frequency of vitamin B12
deficiency than females in Arab and Caucasian populations
\textsuperscript{62}. However, there is some evidence that females have
a higher frequency of vitamin B12 deficiency than
males\textsuperscript{65}. On the contrary, another study revealed a
higher percentage of vitamin B12 deficiency in males (15.2\%) compared
to females (11.5\%)\textsuperscript{66}. Nevertheless, different
findings have reported a lack of relationship between gender and vitamin
B12 level\textsuperscript{60}. Therefore, other risk factors have to be
considered in addition to gender, such as genetic predisposition, and
physiological differences, and pregnancy frequency.

The current findings did not report a difference in the vitamin B12
deficiency between age groups, \textless{} 40 years and \textgreater{}
40 years (\emph{P} =0.119), which is consistent with the results of a
previous study \textsuperscript{62}. On the other hand, most studies
showed a significant association between vitamin B12 deficiency and
age\textsuperscript{54,63,67,68}. In another previous survey that
evaluated vitamin B12 deficiency in Jordan for participants whose ages
ranged between 19 and 90, the study concluded that mean serum vitamin
B12 levels rise with age, however, individuals aged \textless{} 40 years
had the lowest vitamin B12 levels among all ages, which is consistent
with our findings\textsuperscript{16}. Nevertheless, the deficiency of
vitamin B12 may commonly affect the elderly. Therefore, the lack of
association between the age group and vitamin B12 deficiency can be
attributed to the size of the study population from each group.

Herein, the level of vitamin B12 showed a significant association with
the level of Hb (\emph{P} = 0.044). On the contrary, Qutob et al have
shown that there was no correlation between B12 status and Hb
(p=0.545)\textsuperscript{63}. Additionally, we observed a statistically
significant positive correlation between RBCs count and vitamin B12
level (r= 0.354, p=0.001). Metabolically, vitamin B12 is an essential
cofactor that results in normal blood cell maturation. Macrocytosis is a
cause of ineffective erythropoiesis due to vitamin B12 or folate
deficiency\textsuperscript{69-72}. Therefore, our results support the
role of vitamin B12 in the erythropoietic process. Moreover, PCV and
platelet count showed a significant association with the vitamin B12
level in the studied population. On the other hand, our findings did not
show any significant association between the level of vitamin B12 and
the MCV. On the contrary, many studies confirmed the inverse relation
between B12 level and MCV\textsuperscript{63,73,74}. An elevated MCV
refers to the average red blood cell volume that may arise with or
without reduced hemoglobin levels\textsuperscript{65}. Overall,
Pernicious anemia leads to B12 deficiency and results in producing
abnormal and unhealthy RBCs, which means fewer functional
erythrocytes\textsuperscript{75-77}.

The current study also aimed to complete the portrait of the prevalence
of B12 deficiency in Jordan. In a previous study, we evaluated the
prevalence of vitamin B12 deficiency in several regions, starting from
the northern part of Jordan, Irbid (41\%), Karak (35\%), Tafilah (27\%)
and Ma\selectlanguage{english}'an (26\%) where we moved in a southerly
direction\textsuperscript{57}. Here, we reached the most southern part
of Jordan, Aqaba which reported 24.6\% vitamin B12 deficiency. This
study confirms the association between the geographical location and the
frequency of vitamin B12 deficiency in Jordan \textsuperscript{57}. Fora
and Mohammed reported similar observations in the north of Jordan
(48\%), where their study emphasized high B12 deficiency in northern
regions\textsuperscript{61}. Moreover, southern regions had reported
lower B12 deficiency than central regions (Amman), and Hakooz et al
(2006) reported lower rates of B12 in Amman
(48.5\%)\textsuperscript{62}. However, the current results showed a
similar mean level of vitamin B12 in Aqaba's population compared to the
Irbid's population and lower level of vitamin B12 compared to the other
parts of Jordan (Middle-South). Overall, the current study showed that
the deficiency of vitamin B12 in Aqaba is lower than in other parts of
Jordan.

\textbf{Conclusions}

To our knowledge, this is the first study to determine the prevalence of
B12 deficiency in Aqaba province. Vitamin B12 levels were strongly and
significantly correlated with gender and Hb, RBCs, PCV and Platelet
count with non-significant relation to age and MCV. Additionally, the
study confirmed that Aqaba, like other southern regions in Jordan, is
characterized by a lower frequency of vitamin B12 deficiency compared to
the other regions in Jordan. Further studies are needed in other regions
of Jordan such as the eastern regions (Mafraq and Azraq) to complete the
national portrait of the vitamin B12 level in Jordan.

\textbf{Acknowledgments:}

We are very thankful to the Deanship of Scientific Research and Graduate
Studies at Yarmouk University for their financial support to complete
this work. We thank Mr. Adel Rababah for his help in the fine art
production.

\textbf{Conflict of Interest:}

All authors declare the lack of conflict of interest in this work.

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\textbf{Figures legends:}

\textbf{Figure 1.} Frequency of vitamin B12 deficiency in females and
males in Aqaba.

\textbf{Figure 2.} The frequency of vitamin B12 deficiency in different
regions in Jordan (Irbid is representing the most northern part of
Jordan while Aqaba is the most southern part of Jordan).

\textbf{Figure 3.} Heat map of Jordan according to the frequency of
vitamin B12 deficiency. (Irbid, Karak, Tafilah, Ma\selectlanguage{english}'an, and Aqaba)\selectlanguage{english}
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