Vitamin B6 Levels in Cats: Correlation with Age and Body Condition Score

Vy Chu
Vitamin B6
in Cats
Hypothesis
Vitamin B6 levels are negatively correlated
with age and body condition score in cats
What is
vitamin B6?
Essential vitamin
Wide variety of function including
protein metabolism, immune function,
gluconeogenesis, and more
Why does it matter?
Prevalence of vitamin B6 deficiencies in elderly human population
 Lack of data on vitamin levels in cats despite increasing life
expectancy
If and when nutritional interventions may be necessary
Methods
60 cats from cat colony
Age 1-17 years old
Body condition score 4-8
44 females and 16 males
All consuming same diet for all life stages
Blood collection
01
.
Methods
Centrifugated
Plasma removed
Packed red blood cells washed in phosphate
buffer saline
Suspended in triton-X solution to lyse cells
Diluted with phosphate buffer to measure
enzymatic activity
Sample preparation
02
Measuring AST activity as a functional biomarker
for vitamin B6
AST
Vitamin B6 is a cofactor for aspartate aminotransferase (AST)
L-aspartate + alpha-ketoglutarate 
            Oxaloacetate + L-glutamate
Oxaloacetate + H
+
 + NADH         Malate + NAD
+
MDH
AST
L-aspartate + alpha-ketoglutarate 
            Oxaloacetate + L-glutamate
Oxaloacetate + H
+
 + NADH         Malate + NAD
+
MDH
Slope is enzymatic activity of AST
Measure decrease in NADH
Primary activation ratio (PAR)
Activity of AST with added vitamin B6 divided by
activity of AST without added vitamin B6
1 sample has 2 measurements: one without
additional B6 and one with B6
Good indicator of vitamin B6 status
Higher PAR reflects lower vitamin B6 status
Blank has same reagents except alpha-ketoglutarate
BCA Protein Assay
Quantification of total
protein in samples
Enzyme activity will be
expressed in units per mg
of protein
Protein concentration
causes color change
which is measured and
compared to protein
solutions of known
concentration
Calculations
 
Beer-Lambert Law
 
 
A = aborbance
ε
= molar absorptivity
B= length of light path
C = concentration
Solve for concentration
Divide by protein
concentration
Calculate PAR
Statistics
Used JMP for linear regression
Y-variable as activity with no B6,
activity with B6, and PAR
X-variable as age, sex, BCS
Only activity with no B6 and PAR
was statistically significant
Equation for age:
PAR = 4.63 +  0.789xage
What’s next?
Normal Distributed?
JMP analyzed distribution of
PAR values to see if it is
normally distributed
Shapiro-Wilk test: how close
does the data fit to normal
distribution?
What’s next?
Blood samples from VMTH cases
Enzymatic activity and PAR values already calculated for 57 samples
Convert data to normal distribution to identify cut off values for
deficiency
Use to find if any VMTH cases are deficient and if so, does the patient
have any symptoms associated with vitamin B6 deficiency?
References
Limpert E, Stahel WA. Problems with using the normal distribution – and ways to improve quality and efficiency of data
 
analysis. PLoS ONE. 2011;6(7). doi:10.1371/journal.pone.0021403
Marriott BP, Birt DF, Stalling VA, Yates AA. Present Knowledge in Nutrition. London: Academic Press; 2020.
Sechi GP, Sechi E, Fois C, Kumar N. Advances in clinical determinants and neurological manifestations of B vitamin
 
deficiency in adults. Nutrition Reviews. 2016;74(5):281-300. doi:10.1093/nutrit/nuv107
Spinneker A, Sola R, Lemmen V, Castillo MJ, Pietrzik K, González-Gross M. Vitamin B6 status, deficiency and its
 
consequences--an overview. Nutr Hosp. 2007;22(1):7-24.
Ueland PM, Ulvik A, Rios-Avila L, Midttun Ø, Gregory JF. Direct and functional biomarkers of vitamin B6 status. Annual
 
Review of Nutrition. 2015;35(1):33-70. doi:10.1146/annurev-nutr-071714-034330
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Explore the potential correlation between Vitamin B6 levels, age, and body condition score in cats through a study involving blood collection, sample preparation, and measuring AST activity. Understanding Vitamin B6 status is essential for addressing deficiencies and potential nutritional interventions in feline populations.


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  1. Vitamin B6 Vitamin B6 in Cats in Cats Vy Chu

  2. Hypothesis Vitamin B6 levels are negatively correlated with age and body condition score in cats

  3. What is vitamin B6? Essential vitamin Wide variety of function including protein metabolism, immune function, gluconeogenesis, and more

  4. Why does it matter? Prevalence of vitamin B6 deficiencies in elderly human population Lack of data on vitamin levels in cats despite increasing life expectancy If and when nutritional interventions may be necessary

  5. Methods 01 Blood collection . 60 cats from cat colony Age 1-17 years old Body condition score 4-8 44 females and 16 males All consuming same diet for all life stages

  6. Methods 02 Sample preparation Centrifugated Plasma removed Packed red blood cells washed in phosphate buffer saline Suspended in triton-X solution to lyse cells Diluted with phosphate buffer to measure enzymatic activity

  7. Measuring AST activity as a functional biomarker for vitamin B6 Vitamin B6 is a cofactor for aspartate aminotransferase (AST) AST L-aspartate + alpha-ketoglutarate Oxaloacetate + L-glutamate MDH Oxaloacetate + H++ NADH Malate + NAD+

  8. AST L-aspartate + alpha-ketoglutarate Oxaloacetate + L-glutamate MDH Oxaloacetate + H++ NADH Malate + NAD+

  9. Measure decrease in NADH NADH vs time 12 10 8 NADH 6 4 2 0 0 2 4 6 8 10 12 Time Slope is enzymatic activity of AST

  10. Primary activation ratio (PAR) Activity of AST with added vitamin B6 divided by activity of AST without added vitamin B6 1 sample has 2 measurements: one without additional B6 and one with B6 Good indicator of vitamin B6 status Higher PAR reflects lower vitamin B6 status

  11. Blank Blank With B6 No B6 No B6 With B6 y = -0.0002x + 0.5076 y = -0.0009x + 0.7126 y = -0.0093x + 0.5857 y = -0.0061x + 0.7573 0.9 0.7 0.8 0.6 0.7 0.5 0.6 0.4 0.5 0.3 0.4 0.3 0.2 0.2 0.1 0.1 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Blank With B6 Blank No B6 Blank has same reagents except alpha-ketoglutarate

  12. BCA Protein Assay Quantification of total protein in samples Enzyme activity will be expressed in units per mg of protein Protein concentration causes color change which is measured and compared to protein solutions of known concentration Standard curve 700 600 Protein concentration (ug/ml) 500 y = 921.19x - 10.222 400 300 200 100 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 -100 Absorbance

  13. Calculations Beer-Lambert Law Solve for concentration Divide by protein concentration Calculate PAR A = aborbance = molar absorptivity B= length of light path C = concentration

  14. Statistics Used JMP for linear regression Y-variable as activity with no B6, activity with B6, and PAR X-variable as age, sex, BCS Only activity with no B6 and PAR was statistically significant Equation for age: PAR = 4.63 + 0.789xage

  15. Whats next? Normal Distributed? JMP analyzed distribution of PAR values to see if it is normally distributed Shapiro-Wilk test: how close does the data fit to normal distribution?

  16. Whats next? Blood samples from VMTH cases Enzymatic activity and PAR values already calculated for 57 samples Convert data to normal distribution to identify cut off values for deficiency Use to find if any VMTH cases are deficient and if so, does the patient have any symptoms associated with vitamin B6 deficiency?

  17. References LimpertE, StahelWA. Problems with using the normal distribution and ways to improve quality and efficiency of data analysis. PLoS ONE. 2011;6(7). doi:10.1371/journal.pone.0021403 Marriott BP, Birt DF, Stalling VA, Yates AA. Present Knowledge in Nutrition. London: Academic Press; 2020. Sechi GP, SechiE, Fois C, Kumar N. Advances in clinical determinants and neurological manifestations of B vitamin deficiency in adults. Nutrition Reviews. 2016;74(5):281-300. doi:10.1093/nutrit/nuv107 SpinnekerA, Sola R, LemmenV, Castillo MJ, PietrzikK, Gonz lez-Gross M. Vitamin B6 status, deficiency and its consequences--an overview. NutrHosp. 2007;22(1):7-24. UelandPM, UlvikA, Rios-Avila L, Midttun , Gregory JF. Direct and functional biomarkers of vitamin B6 status. Annual Review of Nutrition. 2015;35(1):33-70. doi:10.1146/annurev-nutr-071714-034330

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